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.
Questions related to Regenerative Medicine
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) ?
Copper and Cobalt can improve osteogenic differentiation of mesenchymal cells, but can it be somehow connected with hyaluronic acid?
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?
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?
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?
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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.
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
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?
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:
Leukocytes if possible, with diff
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
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.
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?
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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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.
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.
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.
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
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.
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.
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!
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,
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.
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!
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?
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 ?
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?
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?
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!
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?
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?
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.
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.
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.
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.
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?
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?
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.
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?
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.
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.
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?
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?
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.
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?
The cell proliferation capacity of a cell line is rendered by many factors in in-vivo conditions in comparison to in-vitro.
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?
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!
There are many companies that offer kits (for example ELISA and multiplex) for human growth factors. Please share your thoughts and experience.
Is there any study that shows the ability to add exogenous bioactive molecules as growth factors to plasma concentrates like PRP or PRF?
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
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.
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?
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.
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:
I used different concentrations of 5, 10 and 15%. Literature, however shows higher concentrations to be not effective.
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?
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?