• Use of media and serum in clinical culture of mesenchymal stromal cells
    Last year I was trying to capture and analyze results of all published clinical studies in cell-based regenerative medicine. One dimension that I was able to capture is using of media and serum for mesenchymal stromal cell (MSC) clinical culture. Today, I’d like to share some data. You can read about the methodology of this analysis here. Out of total 116 regenerative medicine clinical studies, 45 used MSC culture. I was able to capture information about media/serum use from 36 […]
  • Antibody array for mesenchymal stromal cell QC

    by Alexey Bersenev on August 27, 2015 · 0 comments

    in mesenchymal

    What do you think of rapid immunophenotyping your cell product on a chip? A group of Japanese researchers described a modified method for high-throughput immunophenotyping of mesenchymal stromal cells (MSC). They used cell binding antibody array with various MSC surface markers and validated it against conventional flow cytometry. The method was described for the first time 10 years ago – it entails:

    Antibody arrays are fabricated by displaying different antibodies on a micropatterned, glass-based chip in a site-addressable manner. The expression pattern of surface markers can be attained by inspecting cell adhesion on every antibody spots for assessing simultaneously reactivity of multiple surface antigens with surface-immobilized antibodies.

    The following antibodies were used in array: CD11b, CD31, CD44, CD45, CD51, CD73, CD90, CD105, and CD254. The authors tested different cell concentrations, seeded onto array and different concentrations of antibodies. Bound cell number became constant at antibody concentration of 125 μg/mL and at seeding cell concentration of 2000 cells/mm2. Data between antibody array and flow cytometry were “fairly comparable”. Slight difference was noted in CD90 expression, where flow cytometry showed 2 subpopulations with high and low expression. Three different cell culture harvest methods were tested. After cell seeding, array was incubated for 15 min at 37C. Unbound cells were eliminated by inversion of the plate before readout.

    Importantly, “It was further found that the density of cells attached to antibody spots was correlated to the mean fluorescent channel recorded in flow cytometry.” So, assay could be used as quantitative.

    It seem to me, antibody array could be a good alternative to flow cytometry. It requires fluorescent microscope for assay readout and less training than flow cytometry. The authors specifically discuss pros and cons of the method in comparison with flow cytometry:

    Importantly, antibody arrays provide a high-throughput method that enable us with analyzing the expression of multiple surface markers at once. This is not always straightforward by flow cytometry even if the latest multicolor flow cytometer is used, although simultaneous expression of multiple markers on an identical cell can be analyzed by flow cytometry. Besides this, compare to flow cytometry, the antibody array-based method has several advantages: Analysis can be performed using smaller number of cells per surface marker and does not require analysts’ proficiency in data interpretation as well as an expensive cytometer. To increase the throughput of analysis, it may be practical to automate some of the analytical process such as determination of bound cell densities.

    What do you think? Will antibody array chips be used in cell products quality control? Will it replace flow cytometry for some applications?

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    Cells Weekly – August 23, 2015

    by Alexey Bersenev on August 23, 2015 · 6 comments

    in notes

    Post updated on 8/26/15 at 5.52 pm EST

    Cells Weekly is a digest of the most interesting news and events in stem cell research, cell therapy and regenerative medicine. Cells Weekly is posted every Sunday night!

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    1. Differential immunogenicity of auto- iPS cell progenitors
    New study, published online in Cell Stem Cell this week, described phenomenon of differential immunogenicity of progenitors, derived from autologous iPS cells. The authors used sophisticated humanized mouse model with “human-like” intact immune system and tested two types of mature cells, differentiated from iPS cell:

    … we demonstrate that most teratomas formed by autologous integration-free hiPSCs exhibit local infiltration of antigen-specific T cells and associated tissue necrosis, indicating immune rejection of certain hiPSC-derived cells. In this context, autologous hiPSC-derived smooth muscle cells (SMCs) appear to be highly immunogenic, while autologous hiPSC-derived retinal pigment epithelial (RPE) cells are immune tolerated even in non-ocular locations. This differential immunogenicity is due in part to abnormal expression of immunogenic antigens in hiPSC-derived SMCs, but not in hiPSC-derived RPEs.

    It is unknown if the same phenomenon applies to human. Also, I’d be curious to see if this differential immunogenicity can be demonstrated by other groups in different humanized models (there are many of them) with different iPS cell-derived progenitors.

    2. Big failures in Phase 3 of clinical trials
    Israeli-based company Macrocure announced this week preliminary results of their Phase 3 clinical trial, using cell product CureXcell in venous leg ulcers. CureXcell consists of allogeneic leukocytes and get injected directly into the skin wound. PR says that “study is not expected to meet its primary endpoint”, and:

    “While we are disappointed with the results of the VLU futility analysis, given the positive clinical outcomes seen in our experience with more than 5,000 patients in Israel, we continue to believe that the technology has clinical merit for patients with chronic and other hard-to-heal wounds,” said Nissim Mashiach, President and Chief Executive Officer of Macrocure.

    Company’s stock is crushed (78% down on announcement).

    US-based company Vital Therapies announced this week, that their Phase 3 trial fails in primary and secondary end points. The company develops cell-based extracorporeal device for liver failure – ELAD®. Vital will stop two trials and re-analyze the data. Company’s stock dropped nearly 30% on announcement.

    3. Results of Tigenix Phase 3 trial in Crohn’s disease fistulas
    Belgian cell therapy company Tigenix announced today (.pdf) preliminary results of their Phase 3 trial, assessing efficacy of local injections of allo- adipose stem cells in Crohn’s disease fistulas. The study met primary end points! Not very dramatic, but significant difference with placebo:

    In the ITT3 population (n=212), Cx601 achieved statistically significant superiority (p<0.025) with 49.5% combined remission at week 24 compared to 34.3% in the placebo arm. In the mITT4 population (n=204), the combined remission rates at week 24 were 51.5% and 35.6% for Cx601 and placebo, respectively (p<0.025).

    Tigenix becomes the first (correction: first allogeneic – see comment) European cell therapy company with successful Phase 3 trial results! Great news for the field! For more details we still have to wait final results and publication.

    4. Human brain in vitro – mass media buzz
    This week, the Ohio State University released a conference report news about achievement in growing human brain-like structure in vitro. It caused a huge buzz in mass media – read some good coverage in Guardian, Wired and NewScientist. Professional peers reacted to this news with great skepticism. The typical reaction:

    Several stem cell researchers WIRED contacted, including one who saw the poster at the meeting, declined to comment on the work. As well they might: The work didn’t say enough to comment on.

    That’s right, no data – no comment! More criticism was expressed on social media – see Knoepfler’s post and tweets.
    While we are waiting for any piece of data to assess, many would agree that it was bad PR move!

    5. Advances in optogenetics
    We are continuing to watch the field of optogenetics with great interest. Two new recently published studies The first report came from Stanford University and describes the first implantable wireless optogenetic device. Implantable device is a big step further:

    We show how three adaptations of the implant allow for untethered optogenetic control throughout the nervous system (brain, spinal cord and peripheral nerve endings) of behaving mice. This technology opens the door for optogenetic experiments in which animals are able to behave naturally with optogenetic manipulation of both central and peripheral targets.

    Seem like the device is so simple and cheap, so you can DIY.

    The second study, published in Molecular Therapy, shows utilization of optogenetics for vision restoration in mice:

    In this study, we solved a major challenge of optogenetic gene therapy for blindness by introducing a native opsin of the mammalian retina, the light-gated GPCR rhodopsin, as an optogenetic actuator, which we found to provide orders of magnitude of enhancement in light sensitivity over the microbial opsin channelrhodopsin. We delivered rhodopsin to blind mice via intravitreal injection of a viral vector containing a cell-specific promoter and showed efficient, cell-specific expression in ON-BCs.

    6. Podcast with pioneers of immunocellular therapy
    Novel Targets recorded a great podcast with “titans” of adoptive cell therapy Carl June (UPenn) and Steven Rosenberg (NCI). I found it very very interesting. Rosenberg is not as optimistic about the future of CAR T-cell therapies in solid cancers as June. Rosenberg’s quote:

    “I don’t think that CARs are going to have much value beyond the hematological malignancies with very few exceptions.”

    Highly recommended!

    7. Patient’s on social media updates impact regenerative medicine trial
    Boston Globe has very very interesting post on social media activity of patients from spinal injury regenerative medicine trial, sponsored by company InVivo Therapeutics. Patients #1 and #2 updates on social media impacted company’s stock shares and their lives (this is a love story!):

    The tweets and the selfies, the uploaded video clips, felt like a natural way for Jesi Stracham to record her halting progress as she fought to recover from a motorcycle accident that had left her paralyzed from the chest down.

    She had no idea, as she tapped away at her iPhone from her hospital bed, what her bubbly posts would unleash.

    The stock of a Cambridge-based biotech company would rise and fall with her updates.

    Jason Napodano, an independent biotech analyst who previously followed the company for Zacks Investment Research, has watched with fascination. “Patients scooping companies on clinical trial data,” he said, “is a new frontier.”

    Yes, this is a new frontier and we have to deal with it! We cannot ask patient to stop their social media activity, but investors and companies execs…. common, guys, do not play such stock games! Be wise and base your judgement on solid data, not on social media updates!

    Highly recommended to read!

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    Fetal bovine serum (FBS) is the most frequently and widely used cell culture supplement in mesenchymal stromal cell (MSC)-based clinical trials. The cost, sourcing, validation of lots and suppliers, associated with FBS are getting more complex with scaling up in later stages of cell product development. Add here questions from regulators about xenogenic ancillary material in your clinical cell culture. Use of FBS could be a major driver of manufacturing Cost of Goods Sold (COGS). Today, I’d like to share some information on FBS and related to it COGS, that I’ve learned from two speakers on recent BioProcessing Summit 2015.

    Fouad Atouf from US Pharmacopeia Convention (USP) presented a piece of unpublished research on development of quality standards for the most frequently used ancillary/raw materials in cell therapy products manufacturing. As the first step, his group made a list of top ancillary materials, used in cell-based therapeutic products, approved on markets worldwide. They analyzed about 30 commercialized cell products, using publicly available information. What material, do you think, top the list? Yes – FBS! Manufacturing of over 50% commercial approved on a market cell products, includes use of FBS. Based on such wide use, USP is prioritizing development of quality standards for FBS.

    Anthony Davies – cell therapy consultant, emphasized significance of high COGS as a factor of commercial failure. He proposes a rule: Contribution of COGS to cell product total cost must be <10% and the cost of the most expensive raw/ ancillary materials must be <10% of COGS. Manufacturing of cell product, which does not fit into this rule, more likely commercially dead in long run. The example that he brought up was Provenge (by Dendreon) - COGS on approval in 2010 were 75% of product manufacturing cost, it stayed flat in 2012 - 75%, finally dropped to 50% in 2015 and now is projected to 25% by 2017. So, Provenge broke all commercially viable COGS rules, as per Davies. One of his advice for developers: "The most expensive unit operation in raw materials - the one that you have to change in further advanced development; and the first candidate - yes, FBS!" He said: "Do not use FBS! Please don’t!"

    I think, this is very good advice. Changing essential raw/ ancillary material in advanced stages of development can cost you new IND submission = lots of money and wasted time. So, try to think ahead, keep your hand on a pulse of latest trends in cell therapy manufacturing and... read this blog!

    ***********************
    This post is a part of Not Lost in Translation online community project. In this series we will try to bridge the translational gaps between scientific discovery in research labs and clinical cell applications for therapies. We will look at challenges in translation of cell product development and manufacturing in academic and industry settings. If you would like to contribute to this community project, please contact us!

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    Cells Weekly – August 16, 2015

    by Alexey Bersenev on August 17, 2015 · 0 comments

    in notes

    Cells Weekly is a digest of the most interesting news and events in stem cell research, cell therapy and regenerative medicine. Cells Weekly is posted every Sunday night!

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    1. Update on fetal tissues donation/trade scandal
    StemExpress – the company, which resells fetal tissues to researches, announced termination of partnership with US Planned Parenthood. From California Stem Cell Report blog:

    “We value our various partnerships but, due to the increased questions that have arisen over the past few weeks, we feel it prudent to terminate activities with Planned Parenthood. While we value our business relationship with Planned Parenthood, that work represents a small percentage of our overall business activity and we must focus our limited resources on resolving these inquiries.

    I’m skipping mass media coverage, but interesting discussion on the subject was posted by top medical journal – NEJM this week. In the first piece, entitled Fetal Tissue Fallout, Alta Charo criticized “sting operation of anti-abortionists to discredit Planned Parenthood and highlights the value of fetal tissue research:

    By using the public’s unfamiliarity with the history and realities of fetal tissue research as a back door for attacking Planned Parenthood, abortion opponents have added millions of people to the collateral damage of the abortion wars. This attack represents a betrayal of the people whose lives could be saved by the research and a violation of that most fundamental duty of medicine and health policy, the duty of care.

    In another piece, entitled Planned Parenthood at Risk, the authors voiced a support for Planned Parenthood in “this hard time”:

    We thank the women who made the choice to help improve the human condition through their tissue donation; we applaud the people who make this work possible and those who use these materials to advance human health. We are outraged by those who debase these women, this work, and Planned Parenthood by distorting the facts for political ends.

    The real discussion took place in comments section, where many MDs were in disagreement with author and NEJM “editorial politics”. I’ve picked one such comment:

    I am so done with the New England Journal and its leftist editorials. My subscription is cancelled as of today. I cannot in good conscience support with my hard earned money media that justifies the not only the selling of fetal tissues, but performing abortions in such a way as to preserve the organs for better prices.

    2. Xenotransplantation of humanized organs
    This story was the most discussed this week (after fetal tissue scandal, of course) in mass media. MIT Tech Review posted a piece about success of US-based company Revivicor (division of United Therapeutics) in xenotransplantation of humanized pig organs into primates:

    “We want to make organs come off the assembly line, a dozen per day,” says Rothblatt.

    “We are adding the human genes to the pig so you have the organ repressing the immune response, rather than have to give a whopping dose of immune suppressants,” says Ayares. By next year, some of the pigs will have as many as eight added human genes. These genetic changes make their organs more compatible with a human body, but the animals still look and act like normal pigs.

    … a transplant surgeon and researcher at the National Heart, Lung, and Blood Institute, in Bethesda, Maryland, says a heart from one of Revivicor’s pigs lasted two and a half years inside a baboon.

    Great article on the progress in the field – highly recommended!

    3. The story behind of the first “rejuvenating plasma transfusions” trial
    Yet another great piece in popular media, I’d recommend you to read is a story behind discovery “circulating rejuvenating factors in young blood”, appeared in Guardian. It is very lengthy article, but absolutely worth reading. Some of my favorite quotes:

    The study was published in Nature Medicine in 2014. Immediately, emails flooded in to Wyss-Coray’s inbox. Alzheimer’s patients wanted infusions of young blood. So did numerous aged billionaires. One, who flies around in a jet with his name emblazoned on the side, invited Wyss-Coray to an Oscars after-party this year. (He didn’t go.) Another correspondent wrote with a more disturbing offer: he said he could provide blood from children of whatever age the scientists required. Wyss-Coray was appalled. “That was creepy,” he said.

    Alkahest’s ultimate goal – to identify the key proteins in plasma that rejuvenate or age human tissues and then manufacture a product that uses them – could take 10 to 15 years. In the near term, the company has another strategy. Earlier this year, the Spanish blood products firm, Grifols, pledged $37.5m for a 45% stake in Alkahest.

    And what then? One enormous obstacle for hopes of plasma therapy is the limited supply. In a rough extrapolation from the mouse studies, Nikolich estimates that the globe’s entire plasma supply would be sufficient for only half a million of the world’s 15 million Alzheimer’s patients. “That means big questions about who gets treatment and who does not,” he said.

    4. Insight into eye gene therapy trial
    Cynthia Fox of Drug Discovery and Development wrote a great piece on the recent study, published in Science TM.

    An historic University of Pennsylvania/Children’s Hospital of Philadelphia gene therapy trial, launched in 2007, restored vision to many Leber’s Congenital Amaurosis Type 2 (LCA2) patients who would normally go completely blind by their early 40s. Although reviving the retina was one key to restoring these patients’ sight, the new imaging study showed the gene therapy also prompted the brain to rewire, strengthening the visual pathway from eye to brain.

    5. New methods and protocols:
    Decellularized human liver as a natural scaffold for liver bioengineering (Sci Reports)
    Neural induction of human MSC using neural-cell derived exosomes (PLoS ONE)
    Direct reprogramming of mouse fibroblasts in neurons by small molecules (Cell Stem Cell)
    Generation of functional cardiomyocytes from human iPS cells (PLoS ONE)
    Dedifferentiation of muscle cells in regenerative progenitor in newt (Nature Commun)
    Rapid method for isolation of MSC from whole umbilical cord (Cell Transplant)
    Neural induction of human fibroblasts via a genetic material-free approach (PLoS ONE)

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    The future of human limb bioengineering

    by Alexey Bersenev August 11, 2015 tissue engineering

    Fascinated by Hugh Herr TED talk, I’d like to discuss future scenarios of limbs bioengineering. Hugh Herr of MIT Media Lab, expanded a concept of nature-driven design to new generation of bionic limbs. So, here comes the first future scenario – integration of neural tissue and electronics of bionic limb. A quote from Herr’s talk: We want to go a step further. We want to actually close the loop between the human and the bionic external limb. We’re doing experiments […]

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    Cells Weekly – August 9, 2015

    by Alexey Bersenev August 10, 2015 notes

    Cells Weekly is a digest of the most interesting news and events in stem cell research, cell therapy and regenerative medicine. Cells Weekly is posted every Sunday night! 1. Repercussions of fetal tissue donation scandal A scandal, related to procurement of fetal tissues by US Planned Parenthood (PP) is unfolding. It is clear that, “sting operation” of anti-abortion activists against PP was successful (see one of many nice summaries here). Reputation of PP is damaged, future funding is under question. […]

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    Translating Cell Therapies: Academic versus Industry model

    by Alexey Bersenev August 7, 2015 conferences

    This is a presentation from my talk that I gave this week at the BioProcessing Summit. In the first part, I’m sharing the latest update on value of academic vs. industry cell therapy trials. It measured as input – a number of registered clinical trials for the last 4 years, and, as output – a number of publications results of clinical studies. Take home message 1 – academics hugely outperform industry by number of cell therapy clinical studies and publications […]

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    Clinical Cell Processing News – part 4, 2015

    by Alexey Bersenev August 6, 2015 clinical lab

    Clinical Cell Processing News is a series about new protocols, products and techniques for clinical-grade cell processing and manufacturing. Cell processing devices, cultureware, bioreactors, GMP-grade reagents, cell separation techniques. This series is posted every 2 months. FEATURED: Validation of fully automated device Stempeutron for isolation of adipose tissue-derived SVF (Stem Cells Int) FREE Cell composition included CD34+CD31− adipose stromal cells, CD34+CD31+ endothelial progenitor cells, and CD34−CD31+ endothelial cells, and their relative percentages were equivalent to SVF isolated by the manual […]

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    Cells Weekly – August 2, 2015

    by Alexey Bersenev August 2, 2015 notes

    Cells Weekly is a digest of the most interesting news and events in stem cell research, cell therapy and regenerative medicine. Cells Weekly is posted every Sunday night! 1. Update from the first iPS cell-based clinical trial As you may know, the first iPS cell-based clinical trial in Japan has been suspended – read here and here. Over the last few days, more details about trial suspension came to the light. This week, New Scientist – one of the first […]

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    Advance your cell, gene and immunotherapies towards commercial success

    by Alexey Bersenev July 31, 2015 conferences

    This is promotional post by Howard Choi Learn to how to apply the cost-effective and efficient strategies you need to advance your cell, gene and immunotherapies towards commercial success by attending IBC’s Cell Therapy Bioprocessing & Commercialization meeting, held September 30 – October 2, 2015 in Alexandria, VA. For full program details, download the brochure today at http://bit.ly/B15188SCA KEYNOTE SPEAKERS Translation of Regenerative Medicine Julie G. Allickson, Ph.D., Wake Forest Institute for Regenerative Medicine Building our Biological Future Rob Carlson […]

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