Science topic

Regenerative Medicine - Science topic

A field of medicine concerned with developing and using strategies aimed at repair or replacement of damaged, diseased, or metabolically deficient organs, tissues, and cells via TISSUE ENGINEERING; CELL TRANSPLANTATION; and ARTIFICIAL ORGANS and BIOARTIFICIAL ORGANS and tissues.
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Induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine and disease modeling. The original methods to grow human iPSCs utilized methods developed for human embryonic cells (ESCs), in which mitotically inactivated mouse-derived fibroblasts are mainly used as a “feeder” cell layer to maintain the undifferentiated status of pluripotent stem cells.
https://www.thermofisher.com/kr/ko/home/references/protocols/cell-culture/stem-cell-protocols/ipsc-protocols/ipsc-cells-with-ksr.html (Thermo Fisher Scientific company have feeder free iPSCs but only for research purpose.)
My question is;
How to buy feeder-free iPSC cell which we can buy and use for business purpose (DIAGNOSTIC USE) ?
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Dear Muhammad,
I am not sure if I understand your question, but,
Pluripotent stem cells (ESCs and iPSCs) can be cultured in feeder free commercially available feeder-free ready to use culture media. For instance, Essential 8 medium form Thermo fisher. The phrase "research use only" indicates that this product is not suitable for clinical application. You can order it because you are going to do some in vitro (diagnostic) tests.
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Copper and Cobalt can improve osteogenic differentiation of mesenchymal cells, but can it be somehow connected with hyaluronic acid?
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According to WHO, "the categories of xenotransplantation procedures include the following:
Solid organ xenotransplantation; cell and tissue xenotransplantation; extracorporeal perfusion ; exposure to living animal-derived material "
What resources or articles would you recommend for their procedures?
Thank you.
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Take a look at this paper Holzer et al. (2020) "Immunological response in cynomolgus macaques to porcine α‐1,3 galactosyltransferase knockout viable skin xenotransplants—A pre‐clinical study" as well as at other papers from the Xenotransplantation journal.
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As you might know, regenerative medicine is an ever-growing field of study and research. In this context, several approaches (ranging from classical scaffold-based to the emerging bioprinting) have been developed to fabricate tissue substitutes.
What are the most essential skills for a novice researcher in tissue engineering?
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I believe the most significant revelation in tissue engineering of the new decade will be the use of mathematics to life-like design and interpretation of outcomes experiments in vivo; material science to the formation of smart scaffolds; and data analysis to analyze the unstructured data.
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Is cell counting an integral part of your cell culturing process?
Do you waste valuable time by manually counting? Are you using an automated method that you just don't trust? Are you worried about the negatives of using Trypan Blue?
Well have you tried the NucleoCounter NC-202 by ChemoMetec? If not, click this link to read all about it: https://www.linkedin.com/smart-links/AQH35FOiPkPD8A
After reading, feel free to email me at rfo@chemometec.com to schedule a trial or get more details.
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Thank you for your feedback John Schloendorn, I could see how you think it is a sales pitch. But thats not the case, rather we are sponsoring key labs across North America by lending them our instruments to use :)
If you know of anyone who would benefit from using our instruments for a decent period of time, please by all means let them know about me.
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Greetings,
My name is Priya, and have PhD in Regenerative Medicine. I am currently based in Australia and hold expertise in stem cells and human platelet rich plasma based tissue engineering.
I would like to know more about the project.
Looking forward.
Thanks,
Kind regards,
Priya
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Thanks priya
I wrote this book chapter named Transfusion update as associate editor, published by jaypee publishers and inaugurated in National conference of ISBTI organized by our department at GMC patiala
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Creating an engineered tissue graft is a challenge - concrete detailed protocols are rare and quite welcomed.
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Dear Shengwen Calvin Li,
Details of the grafting requirement and challenges is presented here.
The human body has limited regenerative capacity in most of the major tissues and organs. This fact has spurred the field of regenerative medicine, promising to repair damage following traumatic injury or disease. Engineered tissue graft is defined as a combination of cells and a scaffold material structurally organized into an in vivo-like tissue that restores form and function to an organ.
Requirements for Grafting
1. Selection of biomaterials- This is first but utmost useful steps. The biomaterials used for engineered tissue grafts vary by the tissue being repaired, the cell incorporation strategy and the required biochemical, mechanical, and degradative properties. Here you have to focus.
2. Second is Bioreactors, which can be used to grow tissues grafts in vitro and are designed to recreate key aspects of the in vivo environment and drive tissue growth. Mechanical loading is one of the primary applications of bioreactors and is widely used. There are another modified bioreactors designed to deliver other tissue-specific cues (e.g., electrical stimulation, oxygen gradients,and growth factors).
3. Integration of the engineered tissue graft with the host tissue is a complex process. The graft implant environment varies depending on the tissue or organ being repaired and the disease state. It is necessary to optimize first the engineered graft to match the clinical performance requirements and unique biomechanical, cellular and molecular profile of the host tissue. This will maximize the ability of the graft to restore form and function and minimize the chance that the graft itself will become diseased (it is possible, possibility are there). Graft innervations challange is a primary concern when the purpose of the graft is peripheral or central nerve repair.
Challenges
What are the main challenges?
The specialized function of most tissues and organs in the body poses unique challenges for the fabrication and implantation of engineering tissue grafts. Vascularization, innervation, immunogenicity, thrombogenicity, optical properties, and mechanical properties are a subset of the tissue properties and host response that need to be considered. In most cases, the tissue and organ specific needs will dictate the biomaterial and cell types used the in vitro or in vivo development protocol, and the implantation strategy. In addition to biomaterials, key factors for tissue engineering therapies include expansion and incorporation of specific cell types into the engineered graft, development/growth of the graft in bioreactors or in vivo, and integration of the engineered graft with the host tissue.
Technology
ECM proteins as biomaterials, new fabrication technologies are needed that will assemble complex 3D scaffolds comparable to cell-assembled ECM. Such technologies in combination with advances in stem cell biology and in vitro bioreactors will improve the structural and functional performance of engineered tissue grafts and enhance integration and subsequent regeneration of damaged tissues.
Ashish
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Buddy Ratner @ University of Washington, US
Sheila MacNeil @ University of Sheffield, UK
Mitsuru Akashi @ Osaka University, Japan
Graca Raposo @ Institut Curie, PSL Research University, UMR144, CNRS, F-75248 Paris, France
Vitor M. Correlo @ Institute of Excellence on Tissue Engineering and Regenerative Medicine, Portugal
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Skin Tissue Models is giving a quite nice overview about currently published and/or commercialised skin tissue respecting different levels of complexity.
ISBN-13: 978-0128105450
ISBN-10: 0128105453
Published by
Alexandra P. Marques
Rui L. Reis
Rogério P. Pirraco
Mariana Cerqueira
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Dear Researchers,
We have generated an inducible satellite cell-specific knockout model (iIpax7-cre). Our aim is to confirm a negative impact of our KO on muscle development. To do so, we are inducing KO at ~8wo and attempting to injure muscle and induce expansion of the target protein-depleted satellite cells using cardiotoxin injection.
Our lab is new to this and we are observing less effect on muscle development markers in our controls following cardiotoxin injection than we would expect. We are aware of significant batch variation issues in the literature/through collaborators. We have read about other muscle injury models.
Aside from this, I am unsure whether this is the best method to address our aim. Could any researchers with experience in the area please provide suggestions?
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Cardiotoxin works but is not my preferred way for inducing a traumatic skeletal injury. About 20 years ago, we compared it against using a freeze injury. The freeze injury was more consistent in the amount of injury induced. The problem with using cardiotoxin seemed to be related to the volume of cardiotoxin injected into the muscle. In our case, we were using the mouse tibialis anterior muscle. When injection volume exceeded a few microliters, there would be leakage of cardiotoxin from the muscle. FYI, I am attaching an article that describes our freeze injury technique. We do use it for studying muscle regeneration.
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I am a physician and am very good at what I do. However, I am equally poor at things I do not do, which in this case is understanding the best type of in office Automated cell counter for my practice.
My goal is to have a small, in office machine, which has the ability to accurately and consistently compare whole blood and bone marrow with post-centrifuged samples in an effort to obtain meaningful information for injectate composition. I am open to any and all thoughts on possible benefits to certain information, as I am limited in even the knowledge of what could/would be available to me in such a machine. ie, I don't want to require the ability to designate CD-44 and CD-90+, if the reality is that this is not feasible in such a machine.
To that end, what I do require is the ability to evaluate whole blood/bone marrow aspirate and centrifuged samples for:
Platelet concentration
Nucleated cells
Leukocytes if possible, with diff
If possible:
Anything which would give me a more accurate determination on MSC % in a sample.
Anything with the potential to determine concentration of Growth factors
Anything concerning the above, which would have a likelihood of helping guide future treatments
Requirements:
Size: Nothing larger than an office copier, but prefer something table top.
Cost: This is more variable, but is a 'bang for my buck' type of situation. Accuracy, reliability, usefulness, and value go hand in hand.
Consumables: I would prefer a 'cartridge' type consumable, but this is purely for ease of use. Again Accuracy, reliability, usefulness, and value go hand in hand.
Ease of use: Something which will allow my technologist to perform the test with minimal difficulty and maximal reliability. This includes everything required to go from a 'syringe with WB to a pipette with a centrifuged sample.
Speed: Needs to be useful within a couple minute time frame. Decisions on the procedure will be made after the results are obtained, so a quick turn around is a must.
This is a large order, and the simple fact that I have written the above is testimony to my lack of knowledge on the subject. But, I would like your opinions and recommendations, if you'd be so kind. Even recommendations on where to go to gain a better understanding of what can be done vs dreams/delusions. Cell counters are everywhere, but knowledge is key in this aspect and I wish to make the best decision I am able, based upon gaining that knowledge from good sources, rather than a flashy video and marketer luncheon.
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Hi,
I agree with Gulderen, the blood counter will give you your blood cell counts. They are not very big and give results quickly. A flow cytometer is more complex and you will need to stain your samples before running them so that means that a lot of other lab instruments are needed which will be available in an average lab. I am not sure if there are kits out there for blood that will allow you to label and run your sample without any washes etch. I am not sure if you can measure growth factors in an automated type of machine. We use elisa plates but that again needs time and other consumables/equipment.
Hope that helps
Tania
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In a patient with hereditary desminopathy (Thr341Pro DES mutation in the heterozygous state), a significant loss of muscle mass is observed after a night's sleep, with its replacement by adipose tissue. How to reduce muscle loss during sleep?
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Dear Ali Javadmanesh, Adrian Fierl, Abdulnabi Abdullamer Matruod, thank you very much for your answers!
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Sphere formation is known among normal or cancer stem cells. But whats the underlying mechanism of cells to come together and make sphere?
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A sphere is the lowest energy configuration for bound system (like a bubble). Other cell shapes require focal adhesion on the outside of the cell or asymmetrically organized structures like actin filaments on the inside of the cell. Cells often temporarily "abandon" the production of some adhesive and structural proteins when changing states to redirect energy to the new process and allow for some reorganization.
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Hi everyone!
ChemoMetec is a strong supporter of the biotech incubator initiative. We want to help them grow so your start-up can grow as well!
ChemoMetec is now offering a special program to bring automated cell counting to the shared lab space of YOUR incubator! Contact me directly if you would like to be considered for this unique program!
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Shamsher S. Kanwar Would you like to send me an email so I can respond with some brochures and demo videos? RFO@chemometec.com
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Immune oncology drug programs are increasingly moving towards use of allogeneic T cells which can be engineered and used with unmatched recipients. Celyad, for instance, recently got clearance for trial of one such product. Cellectis is working on similar approaches. These are moving toward being "off-the-shelf" cell products. But I wonder how scalable these approaches really are. Is it possible, for instance, to isolate/expand enough T cells from just one donor to accomplish adoptive transfer to 10 patients? to 100 patients? to 1000 patients? Or will more frequent donations and small batch processing be needed to make these technologies viable? Thanks in advance to anyone who has insight on this question.
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HLA match is an important issue for allogeneic transplantation of any type of nucleated cell. There is need for a wide collection for "off-the-shelf" use or a method to prevent the expression of MHC on the surface of cells. Also, ways/methods to block mutations have to be standardised as the cells have potential of having mutations in long term culture conditions. Opinion of an immunologist..
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I am a graduate student and would like to know which areas have promising potential for future funding. This information would help me choose professors. Thank you.
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Samrat Das Thank you for our response Mr. Das. More specifically I wanted to know about Caner and Immunology, and which areas of therapeutics would be more beneficial for the future.
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How to identify neurogenesis in adult human retina? Is there any good non-invasive method? This is an ethical, technical and tricky question which may give rise to important concepts. What techniques should be used to achieve this goal.
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It's a contentious question if consider it in human for which there is almost no evidence. In animal models there are certain reports, especially in Zebrafish. Just a lab have published a preprint about it. https://www.biorxiv.org/content/early/2016/06/08/057893
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preservation of neural stem cells (NSCs) consider an important step for future works specially in the field of regenerative medicine, so for who long time we can reserve these cells and is there new protocol for that?
thanks to all
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Just to highlight the importance of keeping the dewar with enough Nitrogen if multi people have access to it. It is very easy to find the nitrogen has gone during the weekend! if no alarm, etc installed.
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In tissue engineering scaffold-free methods rely on cells to generate ECM to support tissue fabrication. Can electromagnetic fields be used to stimulate cells to generate ECM?
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Thank you all for your answers.
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hydrogels are materials that have a high water content and are similar to the natural extracellular matrix. they are used as cell carriers in tissue engineering. in many studies about 3D bioprinting of vessel-like structures, sodium alginate solution  is used as hydrogel and I dont know the reason of this event.
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In addition to the replies by Hatem, Sang and Amin, I recommend the attached book chapter on alginate properties and applications.
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I have made collagen based hydrogel for regenerative medicine applications. Since the growth factors are incorporated into the hydrogel, sterilization process should not affect the stability and integrity of the hydrogel for further processing. Kindly suggest me the method and procedure of sterilizing them.
Thank You
Nisha.P.R
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Hi Nisha
The sterilization of scaffolds with growth factor has always been an issue. the major things that you can try out are
1. prepare the collagen hydrogel in a sterile environment, i.e within a clean hood and add GF 
2. UV sterilization can also be done 
3. ETO sterilization. I had actually compared the effect of ETO sterilization on my scaffolds containing platelet rich plasma. When PRP as such was ETOd i could observe a decrease in growth factor activity. however when the PRP was incorporated in my scaffold and then ETOd, I found the activity was not affected.
However ETO sterilization may change your hydrogel property.
So it would be best to try out the different techniques and ensure that growth factor activity is mot lost as well as your hydrogel properties are not compromised. 
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Hello everyone,
I am trying to make iPSCs by using Yamanaka factors (OSKM). I am confused that should I use human derived factors or mouse derived? At this moment, my target is to use human cells for making human iPSCs. Should I use human derived OSKM (hOSKM)? If yes then will these hOSKM factors also work for mouse cells to make mouse derived iPSCs and vice versa? 
I will be highly thankful for valuable suggestions! 
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Yes, you can use human TF factor in mice or other animals. (We used same ones in sheep pig) 
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Hello! I would like to ask about the quantification of sGAG after the chondrogenesis of mesenchymal stem cells.
My principal doubt is how to extract the sGAG to performs an assay with Dimethylmethylene Blue Assay (DMMB) because I have found protocols for that and they talk about the complex sGAG-DMMB but they do not mention how to obtain the sGAG in solution... Maybe with the application of the reagent the sGAG are realising to the medium as sGAG-DMMB complex directly after following the protocol?
Another issue, if I do Alcian blue/nuclear fast red stain. I cannot use them for the abovementioned assay right? I should have two pellets (one for the histology and one for the quantification)
Thanks a lot,
Sergio.
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Alternatively, you could use an Alcian blue based assay. But be forewarned they stopped making Alcian blue back in 1979 due to its highly carcinogenic activities during manufacture (large number of employees making stain were getting cancers). So any Alcain blue purchased after 1979 is not anywhere near to being 100% stain, it is mostly fillers. Alcian blue assay protocols can be found in:
Young HE, Dalley BK, Markwald RR. Glycoconjugates in normal wound tissue matrices during the initiation phase of limb regeneration in adult Ambystoma. Anatomical Record, 223:231-241, 1989.
Young HE, Young VE, Caplan AI. Comparison of fixatives for maximal retention of  glycoconjugates for autoradiography, including use of sodium sulfate to release unincorporated radiolabeled [35S]sulfate. Journal of Histochemistry and Cytochemistry, 37:223-228, 1989.
Young HE, Carrino DA, Caplan AI. Histochemical analysis of newly synthesized and resident sulfated glycosaminoglycans during musculogenesis in the embryonic chick leg. Journal of Morphology, 201:85-103, 1989.
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Stem cell therapy
Empt nose syndrome
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Hello
Look at this article ( The expansion of autologous adipose-derived stem cells in vitro for the functional reconstruction of nasal mucosal tissue )
Good Luck
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Sattler S, Rosenthal N.  The neonate versus adult mammalian immune system in cardiac repair and regeneration. Biochim Biophys Acta 2016;1863(7 Pt B):1813-1821
The above article is insightful and I believe a key cornerstone to the dream of reparative medicine.  They have outlined the direction we should travel.  The question is how can the adult immune system be manipulated to act like a prenatal system?  This manipulation increases vulnerability to infections, so a strategy is also needed to protect the host from invaders while the immune system is altered during repair.
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Removing memory cells may be a significant step towards getting immune system to star over.
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Dear all,
I am starting an experiment with adipose derived stem cells (ASC), but I would like some help or confirmation for the following issue.
Background: I am harvesting murine fat from the inguinal region. In order to obtain the stromal vascular fraction (SVF), I do the mincing, digestion in collagenase 0.12%, stopping the enzymatic activity with stromal medium, sieving through a 40 µm filter and centrifugation as commonly described. I start my culture in 10 ml DMEM + 10% FBS + 1% streptomycin-penicillin on day 0 in a cell culture dish with diameter 10 cm and surface approximately 78.5 cm². The amount of cells I add to one flask is 5 x 10^5 or 1 x 10^6. The former corresponds to a density of approximately 6 400 cells per cm², the latter corresponds to a density of approximately 12 800 cells per cm². I obtained 80-90% confluency after 4 days, so I counted the cells and replated them. The cell number increased slightly from 5 x 10^5 to 5.3 x 10^5, and from 1 x 10^6 to 1.4 x 10^6. But, at the next replating steps, my cell number gradually decreased or stayed the same.
The question I am asking myself is whether this is related to
1) a decrease in the total cell number, due to unfavourable circumstances, microbial contamination, toxicity of my culture,…
2) a higher purity of the cell culture: the positive selection of ASC, while washing away other (non ASC) non-adherent cells at every replating step
In the first case, I found possible solutions as for example:
- changing the batch of FBS
- increasing the FBS in the stromal medium to 25% (however this may promote premature adipogenesis)
- using α-MEM or DMEM/F12 instead of DMEM
- adding L-glutamine to the medium
- adding bFGF to the culture
- adding ITS (insulin, transferrin, selenium) to improve cell proliferation
- plating the cells in a lower density: between 500 and 4000 cells per cm²
In the second case, I would expect an increase in cell number as ASC should expand clonally.
Because I would like to be sure whether or not I am culturing viable ASC and not degrading / senescent ASC or other cells, I added 5 pictures of my culture dish with attached cells, 4 of them are before and 1 is after washing (during the 3rd passage). I have learnt that once the SVF is plated, the cells that adhere to the surface and multiply are the ASC population, while all other cells are washed away at the first replating step. But is this 100% true? Can we say with certainty that only ASC adhere to the cell culture dish?
Any help would be greatly appreciated in the evaluation of these pictures and the following questions:
- Is this a pure culture of ASC? Or is this still a heterogeneous mixture of several cell types, like fibroblasts?
- Can you draw conclusions about the viability of the cells by evaluating these images?
Thank you, please let me know if any more information is required!
Maxim
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Dear Maxim,
I have to agree with all of the above comments. Rat and murine ASCs are more difficult to maintain in culture than human ASCs for instance, but if you see a decrease in earlier passages, I would suspect something's wrong with your culture conditions (contamination?; mycoplasma?).
I wouldn't go above 20% FBS, you should add glutamine to the medium, platelet lysate does increase cell proliferation but you can get you medium gellified  and you cannot use it for differentiation assays.
FACS is the best tool you have to assure pure sub-populations within ASCs, but if you want to work with the full population of ASCs I wouldn't worry to much about doing it (also the expression of the referred markers varies and most, if not all, are not specific of stem cells or MSCs..only by looking at the combined expression you can withdraw some conclusions).
Good luck with your culture!
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Dear researchers, 
What is your opinion, is it better to use small or large stem cells for therapeutic use, in theory and practice?
Previous findings indicate that i.v. application of MSCs with a diameter ˃25 um resulted in microinfarctions. Does anyone think that the application of large cells is better, for example, for local application, and small cells for systematic application?
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Dear Jasmin
MSCs during culture expanding undergo some morphological changes .Their diameter increse up to 20 micron and in iv adminestration  the majoraty of them will be entrapmented in the lungs.But that is no problem  for systemic adminestration.
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I am interested to check ROS as well as energy level of PBMC of same patient at different time point after Liver transplant ,is it okay i make freez down of PBMC and study ROS as well other energy parameter at the end point of my study?
does freezing change the energy parameter of the cells?
how to proceed my experiment ?
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I don't feel its a good choice to freeze down any samples for ROS estimations..as there maybe significant changes in ROS levels during thawing and processing..also to check energy levels..please try checking ATP, NADH, NADPH and ion levels like Ca2+ etc..i suggest doing them immediately or not delaying it beyond 10-12 h after sample collection when snap frozen cryogenically..
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What do you think about utility of platelet rich plasma (PRP) in treatment of tendon and ligament injuries?
What is your experience with this method and your preferred preparation methods.
Would you expect benefit of such treatment in heamophilia patients?
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Prolotherapy short for Proliferative Therapy has been shown to help heal tendon and ligament injuries with injection with PRP being more effective than Dextrose injection. However, PRP is much more costly.  I have attached an article which is a review of Prolotherapy with an algorithm for treatment.
For Hemophilia patients, there was a study performed  by Buda et al using Bone Marrow-Derived Cells (which is in the continuum of proliferative therapy) infused in the joint during Arthroscopic surgery which showed regeneration and stabilization of joint degeneration.  See reference below:
Cartilage. 2015 Jul; 6(3): 150-5.
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Do children have the ability to regrow their fingertips? If they do, up to what level?
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Depending of the age the small defect of the skin can be healed nicely but as already mentioned in case of bone loss the loss is permanent
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Hi
I need to select some drugs that are important for regeneration and repair. Is there any database that helps me to select drugs based on this criterion?
Best,
Maryam
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http://www.drugbank.ca/ is a drug database that you can search for drug of interest on browse tab. 
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role of iPS Cell in regenerative medicine and their difference from ES cell
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Both are pluripotent cells and can differentiate into different cells but iPS cells are generated in lab after reprogramming somatic cells whereas ES cells derived from inner cell mass (ICM) of embryo before implantation.
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I am a masters student and have a presentation of theoretical applications of regenerative medicine. I need a xenoenzyme to be exocytosed from an endothelial progenitor cell. I thought there would be a know tag/ sequence that could be incorporated to tell the cell to export the protein but I have had no luck finding any. How would you go about ensuring this protein is secreted by the cell?
Any help is much appreciated. Thanks!
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I think you should add at the N-terminus of your enzyme a pre-pro sequence, similar to those of released hormones or growth factors
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I am currently working on tissue engineering, regenerative medicine and orthopedic tissue transplantation. I have used many techniques but to date, I failed to publish my works in those journals with the IF of more than 10. What should I do to increase the scientific impact of my work in order to be acceptable in high impact journals? Does anyone have an experience in this regard helping me to have a good solution for this?
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Also, you may find it really useful to consult a writing instructor, someone who knows about writing research articles in English.
Here is an article that may be useful: 
How to get published in English: Advice from the outgoing Editor-in-Chief
James A. Coleman
The Open University, UK
System 42 (2014) 404–411 
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Are there any disadvantages associated with this process? if there are what are they and how can they be minimised in order to maximise the benefits fo rthe patient?
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i think there are two main disadvantages associated with this promising field of tissue engineering : first is possibility for cells on scaffolds to  get cancerous and detached from scaffold and circulate in blood stream and the second one is lack of control on process of angiogenesis in new engineered tissue which wholly affect functionality of tissue 
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I have been making mouse embryoid bodies (EBs) using the hanging drop method in bacteriological petri dishes for quite some time.
However, recently I have been having this issue where instead of a hanging drop having only one large EB there are 3-4 EBs of smaller size. Is there a possible explanation for that?
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After 2 years of troubleshooting, I have finally come up with a suitable answer to this problem. The answer is "SERUM" quality.
In the past 2 years I have tested, at two separate occasions, about 5 different FBS lots from various manufacturer. Out of the total 10, only two of the serum lots helped me form single large EB in a hanging drop. Interestingly, both the serum, from different manufacturer, were HEAT INACTIVATED.
This was a little contrary to the idea propagated by various books and protocols on the topic of mouse embryonic stem cell culture. Usage of heat inactivated (needless to say tested) FBS is recommended for mESC culture but for making EBs one can use the regular (NON heat inactivated FBS).  
Since it was only heat inactivated serum that gave me the required results I was looking for, I dug a little deeper into literature and realized that heat inactivation not only inactivates complement but it affects the various physico-chemical properties of the serum (http://www.nature.com/nature/journal/v134/n3390/abs/134628c0.html). One of them being VISCOSITY. Heat inactivation increases the viscosity of the serum and it seems that is what is making the single cells come together and form a single EB rather than 3-4 smaller ones.
The changes in the physic-chemical properties of serum on heat inactivation was studied in detail by Dr. P. LECOMTE DU NOÜY in the late 1920s.
The link mentioned below is for his paper published in 1929 where he showed how temperature affects the serum viscosity. 
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I'm planning to evaluate the remedial effects of Mesenchymal Stem cells in glycerol induced acute kidney injury (AKI) so I want to find the right dose of glycerol to establish AKI in rats.
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Hi.. Can you guys give me the relevant references for following this model
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The intra-arterial delivery route of stem cell administration is very attractive in stroke given the direct delivery of cells to injured brain and minimally invasive nature. However, there is no established method to extrapolate rat doses of intra-arterially administered cells to humans. One simplistic theoretical way is to use the brain circulation volume of the two species to extrapolate.
Any suggestions are greatly appreciated.
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You should also take into account different sizes of brain parts. I mean frontal cortex makes larger part of brain in human than in rat, so I guess it will get lager portion of stem cells than in rat but the density will be lower. 
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I would like to build in vitro liver tissue model for drug testing. Since I am freshman in this area, I was wondering is there any techniques for efficiently maitaining functions of hepatocytes? Any answers will be appreciated.
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The culture of primary hepatocytes as spheroids creates an efficient 3-dimensional tissue construct for hepatic studies in vitro. Spheroids possess structural polarity and functional bile canaliculi with normal differentiated function. Thus, hepatocyte spheroids have been proposed as the cell source in a variety of diagnostic, discovery, and therapeutic applications, such as a bioartificial liver. Using a novel rocking technique to induce spheroid formation, kinetics of spheroid formation, cell-cell adhesion, gene expression and biochemical activities of rat hepatocyte spheroids can be tested over 14 days of culture.Hepatocytes can be isolated from 250−300 g male Sprague Dawley rats (Harlan, Indianapolis, IN) by a two-step perfusion method . All harvests yielded hepatocytes with viability exceeding 90% by trypan-blue dye exclusion. Freshly isolated hepatocytes are suspended in culture medium [Williams-E medium with 10% fetal bovine serum (FBS; Mediatech, Herndon, VA), 10 U/mL penicillin G, 100 μg/mL streptomycin, 10 μg/mL insulin, 5.5 μg/mL transferrin, 5 ng/mL sodium selenite] at 1×106 cells/mL. Suspended hepatocytes are inoculated into four different culture vessels: spheroid rocker dishes (10×8×2cm); spheroid rotational flasks (6cm diameter); monolayer plates of tissue culture plastic (60mm; Corning, Corning, NY); monolayer plates of type I collagen-coated plastic (60mm Biocoat; BD Labware, Franklin Lakes, NJ). Cell density (1×106cells/mL) and plating density (1.8×105cells/cm2) are standardized by inoculating cell suspensions of 20mL per spheroid vessel and 5mL per monolayer plate. Spheroid dishes are rocked continuously at 0.25Hz while spheroid flasks are  rotated on an orbital spinner. Monolayer plates are placed on a stable rack. All cultures are  incubated at 37°C and 5%CO2.
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I am working on Human Aortic Smooth Muscle Cells (HASMC). Upon confluence when I change the media of Human Aortic Smooth Muscle Cells (HASMC) and wash with PBS, these cells detach from the surface making sheet like structure, If anyone has experience with these cells kindly let me know how I can overcome this problem and reduce the detachment of SMC during washing and media refreshing.
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Hi, I hope you sorted out cells coming off the flask problem. May I ask what media (and/or supplements) are you using? In our lab we will start working with human aortic smooth muscle cells and human aortic endothelial cells. I am trying to set up a protocol for culturing these cells. However, it seems a variety of different media and supplements are in use for these cells and I am confused which product to buy. Any help will be greatly appreciated! Thanks very much. 
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How can I check whether I can use that or not?
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Hello,
I use routinely human VEGF for my mESC differentiation protocol towards blood and endothelial cells. I use the VEGF from R&D systems (#293-VE-010). I think it should work well on your mouse primary cells.
Good luck.
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Hi all,
I'm working on differentiating ESCs to a forebrain lineage and was wondering how to calculate differentiation efficiencies?  I see efficiencies written in some ESC induction papers (example, some papers quote a 20% differentiation efficiency from their ESCs) but am not sure how these are calculated.
Are they from calculating the number of cells in the final plate (at the end of the differentiation time line) that are positive for a lineage marker vs. the number that aren't?
Example: Final plate is stained with an antibody against a lineage specific protein.  20% of the cells in the field of view (based on DAPI) is positive for this marker.  So efficiency is 20%?
Or is it based on number of starting cells divided by number of cells in the final culture positive for a lineage specific marker?
Example: Start with 10000 ESCs dissociated and plated.  Count number of cells throughout differentiation protocol (assuming that cells are dividing at least initially after plating out, lets say cell number plateaus at 1 million).  After differentiation protocol, end with 10000 cells that are staining positive for the lineage marker.
Would the efficiency then be 10000 (# marker positive cells) /1000000 (cell number at plateau) = 0.01, or 1%?
Or is there some other (probably smarter) way of calculating differentiation efficiency?
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Hello Amy,
You can't count  around 10,000 cells positive for a specific marker  under a microscope and also you are going to compromise your accuracy by same. To be very specific and brief, you can calculate the percentage positivity for a marker which is positive for that partiular lineage by FACS.. Percentage positivity gives you a direct indication of differentiation efficieny. Suppose at control level ESCs are expressing MAP-2 which is a mature neuronal marker, as 0.5% and after differentiation protocol its 95.5% then obviously the efficiency of ESCs to differentiate into mature neuron is 95%. Through FACS you can get a direct record of postive percentage and no need to go through complicated formulas or equations...
Last but not the least, comparision of control and differentiated population is must to get final accurate value of percentage efficiency and your marker of choice should be highly specific for that lineage.
Hope it helps..!
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Would it be reasonable to think that combining Molecular Genetics and Cognitive Robotics will some day (ex. in 10, 50, 100 yrs.) contribute to a successful convergence between man and machine to combat disease and degeneration of humans?
Many science writers seem to say the merger in not too far away now. Should we believe them?
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Biological systems operate through time-dependent adaptive dynamics whereas the architecture of technological systems is exclusively one of central control determining which components perform what functions according to predetermined criteria: the two are fundamentally distinct.
Notice also that genetics describes biological history of adapting to life experiences from a purely molecular-system static (snap-shot photo) perspective (DNA can best be viewed as the substrate by which the adaptive constituents of a biological system develop and, broadly, interact) and tell us nothing about the dynamics of living entities.
Ignore the nonsense coming from techno fan-boys advocating a so-called "critical point" of human-tech convergence; they're merely promoting themselves and, like Hollywood screen writers, ignore the crucial details constraining life (limited energy availability, for one).
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Enamel or Blood?
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Does anyone have a good method for extracting polymer from explanted polymer implants with substantial tissue ingrowth? The polymer can not be physically separated from the tissue, ideally the method would be chemical extraction. Specifically the polymer is poly(lactic acid) or polylactide. I have paraffin blocks of tissue that have been used for histology of the constructs, but I would love to be able to run GPC on the polymer remaining in the block samples. If anyone has any advice please let me know, I am curious how much polymer I will need to extract. Thanks!
I think this question is most relevant for tissue engineering, bioengineering, regenerative medicine, and looking at biodegradable implants in vivo. Tissue could be human or animal. 
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We have used a collagenase digestion to remove fibrous tissue from polymer/tissue explants (see Journal of Surgical Research. Volume 184 (2013) pp. 766-773.)  With this technique, we can recover and characterize the polymer implant by mechanical testing, GPC, SEM, etc.  However, this may be best suited for fresh explants.  In your case, you will likely need to remove the paraffin first.
As an alternative, since GPC only requires a few milligrams of polymer, you could try direct extraction of the paraffin block with a solvent like hexane to remove the paraffin and lipids, followed by your solvent of choice (ex. chloroform) to extract the PLA from the residual tissue.  After extraction of the polymer, you can recover the polymer by evaporating the extraction solvent and then proceed with your GPC experiment.  Note: There are may different forms of PLA that vary in solubility in different solvents, but none of them should be soluble in hexane.
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Can platelet rich plasma be used without platelet activators?
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Dear Priya, I guess your question is about the use of PRP for regenerative medicine such as for topical use, implantology, osteoarthritis. Depending upon the proteolytic activity of the tissue where you apply it, the PRP may eventually gets activated by local thrombin, forms a very weak fibrin gel, activates platelets, and eventually releases a few growth factors. I personally think that this approach of using non-activated PRP may possibly be considered if the PRP is introduced in a cavity (or entrapped by sutures) from which it would not leak; otherwise on opened surfaces the PRP may be washed away. Kind regards, Thierry.  
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Recombinant Human R-Spondin-1 is a vital component of human intestinal organoid culture yet can be ridiculously expensive to buy. Can anyone who performs this routinely recommend a decent, reliable, economic source to buy without compromising on its quality, quantity or activity?
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Hi Rhys,
An economic way would be to get HEK293T over-expressing R-spondin1 cells, culture these cells in basic culture medium (RPMI, 10% serum, glutamine) and collect the supernatant. Then, when you want to culture intestinal crypts to form organoids, use the classic medium (B27, N2, ...) with EGF, Noggin and 5-10% of R-spondin conditioned media. I know at the end the culture medium is not 100% defined as it contains 0.5-1% serum, but it is a limitation that you need to keep in mind.
The other option is to get R-spondin from R&D (good but very expensive), Peprotech (good and works well) or Sino Biologicals (althought the activity is quite low).
Cheers,
Thierry
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PEGDA degredation by MMP sensitive peptides. I want to know if the peptides can be added directly to PEGDA without the peptides being modifyied and what are the reasons for this, if any.
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There are a few points that are missing from this discussion.  If you polymerize a PEGDA with a dithiol (peptide or otherwise) you can react them via two mechanisms.  If you perform a radical polymerization you will get a mixed mode reaction and you'll end up with a gel that is only partially degradable.  If you react them via Michael addition you will only get larger polymers, but will not get a gel at all.  To get a pure, step-growth gel you need thiols-bearing copolymers with a functionality of 3 or more.  So, for example, you could have a peptide that is Cys-MMPsequence-Cys-MMPsequence-Cys, or if making your own peptides you can branch them with a FmocLysFmoc.  But in my experience, these reactions seem to proceed better when the PEG is multifunctional, rather than the peptide. 
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I want to compare the toxicity of a plant based compound on proliferating and non-proliferating cells. For proliferating cells, there are a lot of cancerous cell lines that can be used. Can anyone suggest some cell lines which are non-proliferating excluding primary cells.
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I agree with the idea of immortalized cell line. If you want to go with some simple and inexpensive solution, I would compare sub-confluent and confluent conditions. I suggest to avoid the serum-free conditions, without serum cells stop growing, but the it means a complex stress affecting cellular metabolism. On the other hand, don't add fresh serum (e.g. by changing medium) when you treat your cells.
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We are trying to differentiate the Cardiomyocytes from the human iPSC. We could do 6 differentiation sets properly but suddenly our cardiomyocyte differentiation got stoped we think the only change that we made in our protocol was the change in the splitting ratio of the iPSC. Does the iPSC colony number(Confluency) play such a critical role in the differentiation efficiency?
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Hi Ravindra, I totally agreed with Holger's advise. You better to use Matrigel instead of MEFs and allow the cells become confluent before starting the cardiac differentiation.
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Over the past three decades great strides have been made in the field of periodontal regeneration. Reviews of the literature identify many surgical techniques and materials that have been used successfully to obtain new clinical and histological attachment. Although to date the goal of complete, predictable regeneration has not been attained, the literature has clearly demonstrated the clinical feasibility and histological possibility of periodontal regeneration with many of the procedures. But now the question arises: are those true?
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Dear Saurav
Now there are lot of techniques to assess the regeneration,among which MRI cell viability test which uses gadolinium can tag regenerated tissue.
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I have stored my mouse brain samples in liquid nitrogen just after taking from anesthetized mice. After that I stored them at -80c for prolonged storage. Actually I did not put my samples in OTC nor in 4% formaldehyde as many researchers suggest. Now I have to make a paraffin embedded section from these stored brain (each section cortex, hippo-campus, thalamus separately). Kindly suggest a suitable method to get section and the preferable size of sections.
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Hi,
I would aggree to use that only for biochemistry experiments now. But if you want to do morphology studies, H&E, IF, then you better section the different brain parts before PFA fixation. I would also suggest to carefully freeze the isolated parts in tissue freezing media in isobutanol (cooled down to a milky colour with liquid N2).
Best,
Jonas
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I need to decrease the temperature of homogeneous gelatin solution nearly 0 before adding cross-linker. But I have trouble. Because at low temperatures gelatin becomes solid and I can't add cross-linker. Could you please help me to cope with this problem?
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You have to take gelatin with low molecular weight.
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There are different polyols for fabrication of biodegradable scaffolds. What parameters are important for selection of a specific polyol? Which polyol is the best candidate for both in vitro and in vivo applications?
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Sincerely dear Dr. Matar
I believe that the Science has gained its progress with respect to the great efforts of honourable researchers like you. Surely, devoting time for the synthesis of novel polyesters with unique mechanical properties is worthwhile.
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Is it true in all cases of NPs formulations or not?
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Thanks Daniel.
This is really a new information for me.
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What about iPSCs from mouse neural stem cells by using Oct4?
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It wills show it expression as oct4 is a pluripotent marker. so by using mouse neural stem cells by ipsc.
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To obtain a ideal scaffold for TE one of the most important parameter is the porosity. I would like to know from that value we can consider a structure with high degree of porosity.
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Dear Juliana
Great Question.
As Yang has said, porosity depends on the fabrication and selected materials and their large influence... From my recent experience for one of my customer on making the bobbins, (which is cigarettes outer cover of paper, before stuffing tobacco) porosity is the factor that decides the quality customer experiences, which is largely influenced by pulp, water content, sequezing pressure influencing grain structure etc results in porosity... What we did was to take thro DFSS (design for Six Sigma) approach to freeze the spec on porosity and control spec for significant factors.
Suggest arrive at your spec on porosity that has the best effect on output of the product.
Have great research delivery, surely be with few Innovations
Kindest Regards
Ramanan
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The cell proliferation capacity of a cell line is rendered by many factors in in-vivo conditions in comparison to in-vitro.
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I agree that greater cell numbers are needed for in vivo applications than in vitro. When cells are implanted in vivo, a significant portion (I believe over 75%) of implanted cells will die, so the larger cell number allows for a greater population on the scaffold than a lower cell number that might be suitable for in vitro.
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To overcome the bio-availability and permeability of drugs, nanoformualtions are being developed. Will it be more easy, cost effective and better if the compound can be given as co-therapy with the nano-particles?
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(*Taking Co-therapy as multiple drugs in single administration)
Key technical advances that have enabled the successful development and commercialization of nano-particulate drug products include advances in biology, materials science and particle engineering, processing, and manufacturing.
Nanoparticle selectivity and efficacy is important in parenteral drug delivery and targeting for cancer and other diseases. Also beside nanoparticle selectivity and efficacy, one important factor is their potential toxicity. Further advances in cell and tissue imaging techniques and other in vitro and in vivo characterization methods of nanomaterials and nanoparticles are necessary to better understand and predict toxicity. Furthermore, development and clinical application of multifunctional nanoparticles which combine disease diagnosis with therapy (nanotheranostics) could prove to be very useful.
Pharmacodynamic and Antiretroviral Activities of Combination Nanoformulated Antiretrovirals in HIV-1–Infected Human Peripheral Blood Lymphocyte–Reconstituted Mice.
Upal Roy et. al.
(Attempts have been made to administer the multiple therapy using Nanoformulation)
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Right now, the jury is still out on STAP cells. It is not known for sure if STAP (stimulus-triggered acquisition of pluripotency) is a genuine phenomenon. Top laboratories around the world, in Japan, the United States and elsewhere, are working to try to replicate the findings of Obokata et al. (Nature 505:641-647; Nature 505:676-680). As they do this, I sincerely hope they take the sage advice of Hans Scholer, who suggested that RIKEN (the main team who is attempting to replicate the work) involve Obokata in each and every step of the procedure (see Nature 508:299). Obokata insists that STAP is real. This may be the case, and it may be that the procedure is very tricky; she might have succeeded where others have failed because she has apparently spent every waking moment for years working out the details. This is our (the scientific community's) chance to really get to the bottom of this issue. If we blow this opportunity, we may never know the answer to this important question. If we're not careful, this moment could pass us by.
The reason why it is so important to know if STAP is real is because, if it is, then this will be a paradigm-shifting discovery. In the 1990s (and even earlier, through the work of Sir John Gurdon) we learned that the cytoplasm of an enucleated oocyte can reprogram a nucleus from a terminally-differentiated somatic cell that has been transferred into it (using somatic cell nuclear transfer, SCNT) to a plurpotent state. More recently, in 2006, we learned that a limited set of four factors, the so-called Yamanaka factors, can also reprogram somatic cells to pluripotency, producing iPS cells. These were astounding discoveries, ones that told us something remarkable about the nature of the cell: that it has the capacity to "reverse course" and become a pluropotent stem cell under certain conditions. If STAP is real, then this means that the conditions under which this sort of thing can occur are even broader than we ever could have imagined, to also include external triggers (low pH, mechanical stress). What would this tell us about nuclear-cytoplasmic interaction and the role of the whole cell in gene expression patterns? How would the human body (for instance) normally prevent this kind of reversion to pluripotency, which would potentially lead to cancer? These are exciting questions. Let's get this thing right; let's pursue all avenues to find out what the real answer is!
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My two cents on this subject.
As someone with working experience in and admiration for both plant and animal (mammalian) cells I would like to put things into perspective here. The ability to reverse a 'differentiated' state to an 'embryonic' state was achieved in plant system by simple introduction of plant hormones (cytokinin and auxin) in their culture media. Many mammalian stem cell biologists, sadly, are not aware or do not appreciate this.
Yamanaka's discovery has undoubtedly been a game-changer for biology and medicine but my first reaction to his discovery was, 'its about time mammalian biology caught up to plants.' I felt that perhaps we could do even better.
I am tempted to ridicule the notion of pH induced stress as a method to reprogram but I have been often surprised in my life to know that there exists an inexplicable possibility for it to be real. I am sure everyone is keenly observing the controversy on STAP cells. It is certainly another revolutionary concept (if its real).
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There are many companies that offer kits (for example ELISA and multiplex) for human growth factors. Please share your thoughts and experience.
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Hi Dr. Markert,
I've always had really good results with Millipore's Luminex multiplex kits (assuming you have the dedicated instrument), however, I've really only used them for human/animal serum and cell lysates. Their applications specialists are awesome and can give you really solid advice about whether or not their kit will work. They also have pretty good academic discounts. The only problem, which is the problem with multiplexing in general and not Millipore specifically, is the reagent compatibility. This has always been a limiting factor in how many analytes I can "plex" together and I've never been able to get more than 13 at once despite the theoretical ability to have 100+.
On the other hand, after a bad experience with Invitrogen, now Life Technologies, I swore I would never work with them again, even though I assume they have fixed the issue by now. About 5 years ago I found that I could not get even remotely similar concentrations using the lab's internal control. Basically, we got a stock of stripped human serum and had spiked it with known concentrations of various biomarkers and then subsequently analyzed it with about 5-10 kits from different companies to validate it. the lot of their kits worked fine the first time but then several months later we reordered and they only had a new lot. Suddenly, out control was off by about 50% even though the kits controls were fine. Turned out, their validation procedure only referenced the previous lot, not a reference lot, and only required their validation controls be within 10% of the previous lot. In the intervening months they had made about 15 new lots of this kit and each time the validation was a few percent higher, but less than 10%. The net effect, however, was about a 50% drift. Realizing their error they gave us a full refund on all the kits we had used from them, but it did nothing to replace the wasted human sample from the now dead cancer patients and the 14 months of work analyzing the thousands of samples.
ELISA wise, I really like PeproTech's kits. Their development kits are dirt cheap and I have never had a single problem with their antibodies.
Hope this helps, and sorry for the long story, it still bothers me to this day when I think about it.
-Zack
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Is there any study that shows the ability to add exogenous bioactive molecules as growth factors to plasma concentrates like PRP or PRF?
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They could be used as a vehicle for exogenous growth factors however PRP and PRF concentrates will likely have endogenous growth factor contents such a TGF-beta and PDGF. You might find it hard to characterize bioactivity in response to your exogenous growth factors unless you had very sensitive assays and good controls including the growth factors that could be endogenous to plasma concentrations. I normally use diluted GF from a high concentrated stock in ethanol, diluted in serum free culture medium.
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I have problems in obtaining immunocompromised mice.
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Thanks for ur appreciated reply i'll try ....
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I am working on setting up a protocol for human mesenchymal stem cell intravenous infusion in the brains of ischemic mice. Specifically, I am infusing 11 week old C57Bl/6 mice with 1 million living cells diluted in 500 microliter of saline containing 2 microgram/ml heparin over 1 minute. I have a major problem with the occurrence of a pulmonary embolism right after infusion. Do you have any experience with this? Any tips on how to solve the problem? Many thanks, Elisa
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I suggest to transplant freshly isolated MSCs from bone marrow after FACS sorting. Researchers from Japan published in a recent paper that freshly isolated MSCs that are CD271+CD90+and CD106+ cells do not get stuck in the lung as the cultured cells do. Please check the attached paper.
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I am starting C. elegans research and would like to source some OP50.
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Hi Dan,
Have a look at the CGC (http://www.cbs.umn.edu/research/resources/cgc). They provide lots of nematode strains, but also E. coli OP50 at low costs.
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I am interested to learn and know about adult stem cell research activity and have some ideas for sharing / discussion, but I am not sure about its feasibility so need help for discussion and understanding its feasibility.
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A couple of issues to sort out here.
1. I was not speaking about the kidney because frankly I have not followed the research on it. I felt your original post gave the impression that adult stem cells are not established in most tissues, and I disagree with this completely.
2. The evidence is that the function is defined by the niche. Therefore, to function as a stem cell, a cell must interact with and reside in the niche. Just because a cell can, doesn't mean it does. It is clear in the liver and many other tissues (including tumors) that there are long-lived cells that maintain lineages. I think the mitochondrial lineage tracing work that Nick Wright and his colleagues have performed is the most definitive studies in this area. Therefore, it is not "moot".
3. I would add that in the mammary gland several cell types contribute to gland regeneration, but the entire regenerated gland is a clone as determined by common retroviral insertion sites that develop only after multiple pregnancies in intact tissue. Therefore they are the progeny of a single cell.
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Hepatocytes and Schwann cells are two examples of highly specialised cells that play an important role in regenerative processes - they are not stem cells. Is our obsession with "stemness" a help or a hindrance to future breakthroughs in regenerative medicine?
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This is indeed a very good question and an issue which needs to be discussed in relation to the general "stem cells mania".
Actually, most proposed stem cells based regenerative medicine technologies are not relying on the regeneration of the impaired tissues by the tretment with progenitor stem cells, but on their possible indirect trophic effects, such as secretion of regulatory and growth factors. Actually, others and we have shown the major impact of isolated adult cultured human placental cells implantation for such indirect effects. Those isolated and easily expanded stromal cells which are not "stem cells" by definition still may be well tolerated if even after allogeneic implantation and they could modulate various regenerative processes, such as the bone marrow salvation after high dose irradiation, as shown in our recent publication in PlosOne. These cells also have almost no ability for transformation, which render them safer than other cell therapies. Many have claimed that bone marrow derived mesenchymal stromal (stem) cells (MSC) may have somehow similar effect, but the stemness of these cells, is secondary to their principle indirect effects.
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I would like to do clinical translational research in animal models later on human trials please suggest proven area especially in regenerative medicine or dentistry.
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Hi,
Since you are into dental sciences,may be you can start the work with dental pulp stem cells.They are multipotent but yet have a propensity for the ectodermal lineage and this you can try it for neurological diseases.
Regards
Charan
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I am editor of the Journal of Tissue Engineering: http://tej.sagepub.com/
This is an open access journal published by Sage Publishers. All the papers are free to download. We have progressed well and will be applying for an impact factor later this year. However, with the rapid changes occurring in publishing (particularly the change from the traditional publishing model towards open access, I wondered if there was a way to make the Journal of Tissue Engineering stand out.
We have already had some excellent papers from some of the leading groups worldwide. Some examples here:
and
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Hi Jonathan,
Obtaining the IF is an obvious way (although the fact that J Tissue Eng is indexed by Pubmed offers it the necessary starting visibility) however, in my opinion, given the 'cutting-edge' element of Tissue Engineering research I would opt for invited reviews from researchers that are established in their field which would lead to 'key hits' in search engines. Also, given the fact that you are based at UCL and you have such a strong background in TE and biomaterials I would utilise consortia such as the IBME and the potential for interdisciplinary projects that may arise from UCL collaborations to encourage submissions to J Tissue Eng (from short communications to full-blown articles), coupled with a 2-3 week review process that would be complemented perfectly by the open access format.
Please keep up the great editorial work, the field of TE is in need of more platforms for exposure!
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Is it possible to get the GFP labelled cells on histology 5 months after transplant?
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There isn't an easy answer to this question. In the case of cells labelled through introduction of a GFP gene into the DNA of the host cell in a whole organism context such as transplantation, then a lot depends on the promoter being used. Promoter silencing may take place but with correct promoter choice is not inevitable. GFP is associated with slight toxicity in some cell types and situations, and may also be immunogenic although this varies with the host. But nonetheless it has been used to look at cell fate in a variety of developmental and transplantation models, some of which are fairly long term. One possibility to minimise long term loss of GFP reporting in a transplantation model might be to consider an inducible system.
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I used different concentrations of 5, 10 and 15%. Literature, however shows higher concentrations to be not effective.
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except putting higher concentrations, maybe lowering the difference between concentrations would be good idea. so, as Elbay wrote, at least 20 and 25 should be done to see if 15 is optimum among 5 cases. maybe 17, 19, 21, 23, 25 is optimum, or as Elbay wrote, do it til you see drop or straight line. you should be cautious between optimum and maximum, especially if we speak about resources spent to achieve certain goals.
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We would like to induce osteogenesis and adipogenesis in our hMSC lines, but in addition to traditional qualitative staining (i.e. Oil Red O, Alizarin Red, etc), we would like to analyze the degrees of differentiation by flow cytometry. Is anyone knowledgeable about how this can performed and perhaps what markers can be assessed?
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Hi please take a look at this article for adipo:-
regards
Charan
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I am looking for type 1 collagen that could be used for human translation or anything that is GMP manufactured/medical grade.
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There are actually several companies producing and manufacturing Type I collagen that is used regularly in commercial medical devices. They don't sell gels, but you can easily make a collagen gel from the micronized Type I collagen they do sell. Off the top of my head, I'd suggest Kensey Nash (http://www.kenseynash.com/kensey-nash-technologies/collagen-technology) and Collagen Matrix Inc. (http://www.collagenmatrix.com/).
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What is the best seeding density for MSC and AC in scaffold?
Is 1 million per cm3 ok?
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Dear Jorg,
Many thanks for your answer :) yes I'm from Freiburg and I asked also already from different labs :) but I'm looking to know what other people do all around the world :) to see and check a bit out of box.. I checked my cooment there was typo we culture msc with 3000 per cm2, i think i need to talk again with my lab members and also our collaborators in Basel we culture Chondrocytes in 5000 cell per cm2 in flask, which is 4 times less than what you guys are using..So maybe I need to seriously talk with our guys..
with Chondrocyte I got same answers from almost every body for culture in 3d..my main problem is MSC ! Because I think with seeding high density I will definitely effect contact inhibition growth of MSC and probably they will trigger to differentiate .and I don't want this.. I just want to see pure effect of scaffold surface and properties
So I don't know what seeding density will work for me!! The problem is every body is telling me try to test different seeding densities and so on..but because I have limited number of scaffolds so I really need to do re my best.. And hesitate try and error as much as I can..
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I'm looking forward to culture MSC and AC on my modified scaffold. My problem is that, I do not know what is the appropriate density for culturing MSC or AC on scaffolds. For example I have found that people culture 10^6 cells in 24 well plate size scaffold, but I'm wondering this huge number of cells will not effect MSC's properties, since they will be so close to each other.
For flask culture we use protocol 3000 MSC per cm^2 and 5000 Ac per cm^2.
Is there any relationship between these densities and safe density for cell culture on scaffolds?
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Hi Melika,
You need to consider a number of things.
1) When you say you seeded 30,000 cells, were these viable cells as determined by Trypan Blue exclusion assay, or did you simply count them without Trypan blue? If you did not use Trypan Blue then there is a possibility that the proportion of viable cells is below 30,000. I'm sorry if this seems like an obvious error, but it is not an uncommon one.
2) It is not sufficient to seed the cells on the scaffold alone. Did you use a positive control for the cells (such as tissue culture plastic of a 24 well plate, or Thermanox discs). The initial seeding density may not be the cause of the problem, but the scaffold itself. For example it's biocompatibility, or the method you employed to sterilise the scaffold. A positive control would help establish this.
3) You say that you think you had 0.5 million cells after 3 weeks. Did you harvest and count the cells?
From experience, I know it is not always easy to harvest and recover all the cells from the scaffold, especially after prolonged periods of incubation. Therefore, this might be another issue you need to address, if it is not optimal it would have implications on subsequent analyses.
4) Finally, I appreciate that your aim is to establish the proliferation rate, protein and gene expression, but you may wish to consider examining the cells under SEM to see how their morphologies compare when grown on the scaffold and positive controls (Thermanox). This will flag up any scaffold-related issues.
Good luck
Basil
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What are the institutes for tissue engineering research in India?
Which is the best one?
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I would like to know what the highest volume of bone marrow that can be drawn is. Also what are the sites to draw from in adults. I know the iliac crest is the typical site, but are there others? I have heard that the femer is also used.
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The percentage of progenitor cells declines as the volume aspirated at a specific site in the marrow increases. Practically, the optimal volume per site is about 8-10 ml. Larger aspirates are hemo-diluted with blood. The trochar can be advanced a few mm 3-4 times after puncturing the periosteum and cortex and a separate aspirate obtained after each "advance".
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