Regenerative Medicine - Science topic

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.

Also check https://pubmed.ncbi.nlm.nih.gov/26915044/

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.

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.

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.

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

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.

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.

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.

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

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.

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

Dear Ali Javadmanesh, Adrian Fierl, Abdulnabi Abdullamer Matruod, thank you very much for your answers!

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.

Shamsher S. Kanwar Would you like to send me an email so I can respond with some brochures and demo videos? RFO@chemometec.com

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..

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.

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

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.

Thank you all for your answers.

In addition to the replies by Hatem, Sang and Amin, I recommend the attached book chapter on alginate properties and applications.

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. 

Yes， you can use human TF factor in mice or other animals. (We used same ones in sheep pig) 

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.

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

Removing memory cells may be a significant step towards getting immune system to star over.

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!

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.

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..

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.

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

http://www.drugbank.ca/ is a drug database that you can search for drug of interest on browse tab. 

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.

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

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

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 

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. 

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2323722/ 

Hi.. Can you guys give me the relevant references for following this model

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. 

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.

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. 

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.

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..!

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).

It may be usefull http://www.jendodon.com/article/S0099-2399(14)00962-5/abstract

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.

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.  

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

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. 

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.

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.

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.

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

You have to take gelatin with low molecular weight.

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.

Thanks Daniel.

This is really a new information for me.

It wills show it expression as oct4 is a pluripotent marker. so by using mouse neural stem cells by ipsc.

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

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.

(*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)

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).

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

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.

Thanks for ur appreciated reply i'll try ....

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.

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.

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.

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.

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

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!

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.

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.

Hi please take a look at this article for adipo:-

regards

Charan

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/).

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..

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.

This publication might be of interest to you: http://onlinelibrary.wiley.com/doi/10.1002/(SICI)1097-4644(199702)64:2%3C278::AID-JCB11%3E3.0.CO;2-F/pdf

Good luck

Basil

IIT kharagpur-smst

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".