• Cryopreservation of mesenchymal stromal cells can attenuate clinical immune effects
    As Jacques Galipeau reported in conferences and in the paper, cryopreservation could negatively affect therapeutic “immunomodulatory value” of mesenchymal stromal cells (MSC). There was no independent confirmation of Galipeau’s findings, and many MSC product developers remained skeptical. This week, Katarina Le Blanc published a report, which supports Galipeau’s conclusions and provides more insight into potential clinical value of this phenomenon. Let me just say – this paper could change the field! Le Blanc concluded that freeze-thawed human MSC compared to […]
  • Annual IBC Cell Therapy Bioprocessing meeting has finished this week in Arlington (Virginia). Today I’ll share some information, that I’ve learned from a session, dedicated to cell therapeutics biopreservation and loogistics.

    At the beginning of the conference Lee Buckler (Cell Therapy Consulting Group) highlighted one of the future trends in cell therapy – we will see variety of cell delivery formats (likely not in LN2!). To his point, Aby Mathew (BioLife Solutions) called to think about cell storage and delivery format in broader term – “biopreservation“, rather then “cryopreservation”. Biopreservation includes ambient storage, hypothermic storage, cryopreservation, vitrification and anhydrobiosis (drying). Is era of cryo- LN2-free storage and delivery coming to cell therapy field? Well, at least hypothermic storage is expanding now (vitrification and anhydrobiosis are not yet validated for clinical use). For example, HypoThermosol – cryopreservation-free, cold chain supply solution – is being used in >130 clinical trials. It allows short-term cold storage for few days without significant drop in cell viability and functionality.

    Mathew is known proponent of concept for assessment of delayed onset cell death (learn more from these videos), which shows difference between perceived viability and true viability. Even though true viability measurement (24h in culture after thaw) is more accurate assay for cellular products, we still cannot track cell viability in human body after infusion. In product and process development he recommended to use variety of methods to assess “cell health”, such as: mechanical (attachment), proliferation, engraftment, protein/ cytokine production, cytotoxicity… and try stay away of traditional “trypan blue” test.

    Aby Mathew noticed that: “Billions spent annually on cold chain logistics of biologics, cells, organs”, only because we don’t know good practices and do a lot of errors. There is a Good Distribution Practices (GDP) in biologics logistics, that we can learn and follow. We can implement real-time monitoring of cell product shipping, instead of retrospective “failure analysis” (after product was destroyed). Even simple switching from home-made cryo- and bio- preservation solution to commercially available, will allow to drop a number of errors.

    Another way to avoid errors in logistics and cold chain is standardization and development of new tools. This is a mission of relatively new company in the field – BioCision. The company is entering cell therapy with tools, enabling standardization of cold chain logistics – packing, shipping, clinical site handling. They presented the first case study in cell therapy, where LN2-free, controlled rate freezing container CoolCell was used for clinical trial by TxCell SA. Hazard-free (no alcohol as in Mr. Frosty or LN2 required) freezing process is significant step forward! BioCision also presented some new products: BioT Temperature Stability Systems.

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    IBC Cell Therapy Bioprocessing 2014 – From 2D to 3D

    by Alexey Bersenev on September 17, 2014 · 0 comments

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    Annual IBC Cell Therapy Bioprocessing meeting has finished Yesterday. It was great meeting for me! I’d highly recommend it to everyone, who involved in different aspects of clinical cell manufacturing. The common theme of conference was transition of clinical cell culture to 3D settings. The field is coming to realization that only 3D cell culture will allow efficient industrial scaling up for many clinical applications. For some areas, such as manufacturing of allogeneic cells and bioengineering of thick tissue constructs or organ prototypes, 3D era is inevitable! For example, Athersys (reported by CSO John Harrington) calculated that industrial production of their stem cell product MultiStem, will require production from few trillions (for GVHD) to few hundred trillions (for stroke) per year. Even though, their current process (2D) can get biolliions of cells, it’s too labor-intensive (72 10-layer cell factories per lot require up to 13 technicians on seeding and harvest days) to make it affordable. Another interesting calculation was presented by Mark Szczypka (Pall Life Sciences) – he said: “In order to generate 250B mesenchymal stromal cells you need 1332 10-layer stack cell factories”.

    In order to scale up to billions of cells we have to achieve exponential growth. Flat 2D cultures gives us linear growth, but 3D in suspension can give us exponential cell growth. That means transition to suspension culture should be made for adherent cells (traditionally cultured on flat surface), such as mesenchymal stromal cells (MSC). In the last few years a lot of work has been done to validate MSC culture on microcarriers in bioreactors. Based on recent achievements, we can confidently say that MSCs expansion in suspension on microcarriers is ready to move from process development stage to clinical production. Athersys invested 7 years in process development for scaling up manufacturing with lowering cost of goods. They were able to achieve good results with SoloHill microcarriers. Cell concentration at harvest day was ~0.5M/ml in 2% FBS media and ~ 0.24M/ml in serum-free media. John Harrington (Athersys) says that they didn’t see significant change of product cell characteristics (including potency) while moving from 2D to 3D. Celgene is also validating microcarriers for their placental MSCs. They got very good results with Cytodex beads. So, companies, which manufacturing therapeutic adherent cells (Athersys, Celgene) are moving to 3D suspension cultures. Pluristem made this move while ago, using their own bioreactor. Speakers from Athersys, Celgene and Pall noticed that many things should be considered in process development in transition to 3D. For example, some bioreactors don’t perform well with microcarriers and cells prefer certain type of microcarriers (Celgene was not able to grow their placental MSCs on SoloHill beads). Also, I noticed the absence of good reliable assays for detection of residual microcarriers in final product. If you do transition from 2D to 3D culture system in clinical cell manufacturing, pay attention to product comparability (before and after). Potency assay should be prioritized. Remember, it’s a huge process change and you may end up with completely different product after such transition. So, better to do it early on.

    Let’s switch gears and look at organ bioengineering. Creating of thick tissues and organ prototypes is possible only in 3D. The major missing part here is cell number and cell density. Such constructs will require billions of cells. Jeff Morgan from Brown University reported a new way of manufacturing building blocks for bioengineered organs. He said that knowing how many total cells in our body (~37 trillions), cell number per organ and cell growth kinetics in culture (he mentioned achievable 20-fold expansion every 4 days), we can calculate time for “growing whole organ cells”. Thus, growing cell mass for whole heart or kidney will take ~35 days, liver ~ 40 days. His lab created non-adhesive molds (now commercialized by MicroTissues), which allow cells self-assembly to microtissues at high density (2.4 x 10e8/ml). They designed micro-molds of different shapes, allow to form cell spheroids, toroids (doughnut-shaped) and honeycombs. Few honeycombs can form large honeycomb with few millions of cells. Then they developed a device, which will grip and place honeycombs or toroids on each other analogously to additive manufacturing. These parts can be fused in one construct within 48 hours. These are building blocks for organs! About 1000 large honeycombs will be required to build whole heart. But it’s totally possible! Despite many challenges (vascularization is one of the biggest), we have now some manufacturing tools to build thick tissues and organ prototypes.

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    I’m attending annual IBC Cell Therapy Bioprocessing meeting in Arlington. One of the major highlights of the first day for me was understanding the importance of “democratization” and affordability of therapeutic cell product manufacturing. Lowering the cost of cell product is a key to commercial success. If cost of cell product is very high, it will not be cost-effective, it will not be reimbursed, it will fail clinical adoption. So, in cell product development we must try very very hard to bring cost down!

    Everybody was talking about automation as one of the best ways to bring cost of cell manufacturing down. Stefan Miltenyi (Miltenyi Biotec) said that “cell processing has to become less expensive”. He thinks that “automation is able to resolve many critical issues, related to manufacturing of autologous cell therapy products”. His company did a lot for development of cell processing automation, including the most recent multifunctional device CliniMACS Prodigy. He was talking about possibilities of combining Prodigy with clinical-grade sort-on-chip device MACSQuant Tyto.The company now is making GMP-grade antibody for Tyto.

    Jon Rowley (Rooster Bio) used a term “democratization of cell technologies“, which I really like. He gave a great example of lowering cost of mesenchymal stem cells (MSCs) by implementation of automation:
    Cost per million clinical-grade MSCs, expanded in: T-flask = $100-500, multi-layer vessels = $10-50, suspension bioreactors = $1-5.
    The problem of growing “high cell mass” at low cost significantly amplified in research labs, which build prototypes of tissues and organs for pre-clinical studies. Such labs need to grow billions of MSCs and pay “crazy money” for it (example: 1B of MSC may cost you $100k in T-flask and $10k in CellSTACKs). If such tissue constructs will work, the only way to go to clinic is automation via using suspension bioreactors.

    There is a good progress in development of suspension bioreactors and using microcarriers for MSCs expansion. For examples, PBS Biotech was able to achieve concentration of 3 million per ml in their vertical wheel bioreactors. Increasing cell concentration without compromising quality of the product, will allow to cut media usage and shorten the time of culture. Li Ren from Celgene just reported about generation of 1.2 trillions (!) of allogeneic placental MSCs per one process, using microcarriers in bioreactor. If you have to deal with high MSCs numbers, this is the only way to go in industrial manufacturing.

    So, we can see huge investment and development in “cell therapy manufacturing toolbox”. This “toolbox” will enable successful translation of cell therapies. As Jon Rowley said, “we are in a phase of rapid advancement of cell manufacturing technologies”. So, we should take advantage of these advances to bring cost of manufacturing down.

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    Cells Weekly – September 14, 2014

    by Alexey Bersenev on September 14, 2014 · 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!

    Next week, I’ll live tweet from annual IBC Cell Therapy Bioprocessing conference. Follow me and the conference via hashtag #IBC_CTB14.

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    1. First patient received iPS cell-derived product in Japan
    Friday, September 12, 2014 will become one of the most important landmarks in the history of stem cell research and therapy! This day, the first patient has received iPS cell-derived therapeutic product in Japan. A woman in her 70s, with macular degeneration underwent transplantation of retinal pigment epithelium cell sheets, derived from autologous iPS cells in RIKEN. David Cyranoski of Nature reports:

    In a two-hour procedure starting at 14:20 local time today, a team of three eye specialists lead by Yasuo Kurimoto of the Kobe City Medical Center General Hospital, transplanted a 1.3 by 3.0 millimetre sheet of retinal pigment epithelium cells into an eye of the Hyogo prefecture resident, who suffers from age-related macular degeneration.

    The patient “took on all the risk that go with the treatment as well as the surgery”, Kurimoto said in a statement released by RIKEN. “I have deep respect for bravery she showed in resolving to go through with it.”

    My warmest congratulations to Masayo Takahashi‘s team! Bravo Japan!

    2. More STAP news
    STAP saga is not over guys! A lot of things happened this week – very important things! First, Retraction Watch released reviews of STAP manuscripts, submitted to Science magazine and rejected by August 21, 2012. It triggered lively discussion. Second, day later, Science (via ScienceInsider) released reviews of STAP manuscript from Nature, dated April 04, 2013. Interestingly, all 3 Nature reviewers were negative and highlighted many concerns with manuscript. Nevertheless, editor made decision to “give it a try” and asked the authors to respond to reviewers comments, instead of rejecting it outright. About 8 month later, STAP paper was accepted! Ultimately, it was Nature’s editor decision. So, some anonymous sources leaked STAP papers reviews. There was no official statement from Science or Nature on this. Both leaks are making very compelling case for necessity of open peer-review:

    Why did Nature have to re-review the same paper, without seeing the reviews at Science (apparently also rejected and possibly reviewed by Cell)? Would Nature really have published the STAP papers if the reviews were visible? Would there have been a scandal if the STAP papers were published along with the accompanying reviews that we just saw? Why are we as scientists wasting our time re-reviewing papers before and after publication? Why is there the misleading stamp of approval and quality when a paper is published in the fancy journals? And why do we always have to wonder if it’s in fact a good paper or one of the Arsenic/STAP-type “who knows why it’s published here?”

    Third puzzle is update of STAP cell generation protocol from Vacanti’s lab. Despite the retractions of both STAP papers and related protocols and formal agreement of Vacanti for retraction, he continues to believe in existence of these elusive cells. From Nature News Blog:

    “In recent months, our lab decided to re-explore the utility of a low pH solution containing ATP in generating STAP cells,” Vacanti writes in the revised protocol. “We found that while pH alone resulted in the generation of STAP cells, the use of a low pH solution containing ATP, dramatically increased the efficacy of this conversion.
    “We made a significant mistake in our original declaration that the protocol was ‘easy’ to repeat,” the protocol continues. “This was our belief at the time, but it turned out to be incorrect.

    Are you eager to try STAP 3.0 protocol?

    3. Insurance for regenerative medicine products manufacturing in Japan
    Japanese press reported about new law, which will enforce in insurance of errors in regenerative medicine products manufacturing. It will be effective in November.

    Sompo Japan Nipponkoa Insurance Inc. plans as early as next month to sell regenerative medicine insurance products that would pay up to ¥500 million in benefits. Tokio Marine & Nichido Fire Insurance Co. and Mitsui Sumitomo Insurance are also planning to introduce similar products.

    Introduction of such insurance as means of protecting patients from cell product manufacturing errors, will accelerate widespread adoption.
    The wave of new companies, to which hospital will outsource cell manufacturing will rise soon:

    Hospitals have thus far carried out both treatments and cell cultivation. The outsourcing, therefore, will likely enable regenerative treatments to be conducted more efficiently.
    Also in November, the Health, Labor and Welfare Ministry will introduce a new system to give early approval for the sale of cultivated cells and other products for regenerative medicine.

    4. Tools for scaling up mesenchymal stromal cells culture
    Democratizing Cell Technologies blog has two great posts about scaling up of mesenchymal stem cells (MSC) production. In the first post, they overview new available tools for industrial MSC manufacturing – bioreactors and microcarriers:

    PBS Biotech had a poster showing hMSC expansion in their vertical wheel bioreactors.

    These high cell densities are a significant milestone for the field, as it is the first report (that I know of) of consistently greater than 1 million cells/mL over multiple runs, and with the potential to get to >3 million cells/mL.

    The second post is about cost of goods and some other cell therapy-related talk from recent BioProcess Summit in Boston. On of amazing things they described from talk of company Janssen:

    Like all good PD campaigns, they had quantified goals which were to 1) increase bioreactor yields by >50%, 2) increase downstream process recovery by >75%, and 3) decrease serum by 70%. All of this would dramatically increase the yield from every run and help reduce the CoGs of the overall process. Impressively, Dr. Kamaraju reported that they scaled up to 1000 L single use bioreactors and were able to consistently achieve yields of >300 Billion (3 e11!!!) cells per run.

    I’d highly recommend both posts!

    5. Targeting leukemic stem cell via glucose metabolism
    Selective targeting of leukemic stem cell without damaging of normal hematopoietic stem cells has been a very difficult task. This week, David Scadden’s lab published a study, which demonstrates a possibility of such selective targeting:

    This finding suggests that fine-tuning the level of glycolysis may be explored therapeutically for treating leukemia while preserving HSC function.

    From the news coverage:

    “Cancer cells are not like normal cells in a lot of ways, but one … is that they get locked into a particular way of behaving,” Scadden said. “These cells are so singular in the way they handle glucose that they create a unique opportunity to intervene. Normal cells don’t get so disrupted because they have other energy mechanisms in place.”

    Private companies have been developing drugs that target cancer metabolism, but primarily in solid tumors. Scadden hopes that this study can open the door to industry partnerships and the generation of new treatments.

    6. Ground-state pluripotency in human
    Two recent studies described a new receipts for generation of so-called “naive” or “ground-state” human pluripotent stem cell. First, Rudolf Jaenisch lab proposed to use “kinase inhibitors that induces and maintains OCT4″. Second, Austin Smith’s team used short-term expression of two factors – NANOG and KLF2, to generate “ground-state” pluripotent cells.

    7. Injectable niches for lowering therapeutic cell dose
    You may have heard a lot about injectable hydrogels in regenerative medicine recently. One of the reasons to use hydrogels as cell carriers is lowering therapeutic cell dose. Recent study has demonstrated feasibility of such approach:

    Dramatic improvement in cell retention, survival, and therapeutic effects enabled by the primed 3D microniches was demonstrated in treatment of critical limb ischemia (CLI) in mouse models compared with the free cell-based therapy. To the best of our knowledge, this is the first convincing demonstration of injectable and primed cell delivery strategy realizing superior therapeutic efficacy with the lowest cell dosage for treating CLI in mouse model.

    8. New cell therapy blog
    Please welcome in – CellTherapyWonk. This blog was just started by Mark McCall. Mark is an expert in “Cost of Goods” in cell therapy manufacturing. He recently got PhD from Center for Innovative Manufacturing in Regenerative Medicine of Loughborough University.

    Highly recommended!

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    bonus:

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    Cell Gadgets Review – 2014 part II

    by Alexey Bersenev September 12, 2014 gadgets

    RegenMed and Cell Gadgets series is an overview of “smart devices”, biochips, matrices and biomaterials for research and therapy. FEATURED: Low cost high resolution 3D bioprinter from BioBots! 1. Remote control of engineered photo-switchable cells Can you imagine a possibility to control of spacial organization 3D microtissues in culture remotely? It’s blowing my mind guys, because it’s possible now! Group of researchers demonstrated that engineering of cell surface can provide a remote control to on-demand assembly and differentiation of cells […]

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    Isolation of hematopoietic stem cells by single marker

    by Alexey Bersenev September 10, 2014 hematopoietic

    Hematopoietic stem cells (HSC) is the first type of adult stem cells, which was prospectively isolated by defined markers. Characterization of mouse and human HSCs and their isolation by FACS have gotten more and more sophisticated with years. Now, it typically includes combination of 6-9 surface and “functional” markers. But attempts to identify unique HSC marker remain elusive. Recently, Derrick Rossi’s lab from Harvard University described unique single marker for mouse HSC – Fgd5. They started from mining the data […]

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    Cells Weekly – September 7, 2014

    by Alexey Bersenev September 7, 2014 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. Is autofluorescence the answer for “STAP phenomenon”? Japanese media outlet Nikkei reported possible explanation of recent failed attempts to reproduce STAP protocol by RIKEN team: According to the report, which was released by project leader Hitoshi Niwa and other Riken officials on Aug. 27, what looked like clusters of cells could […]

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    Is using platelet rich plasma a cell therapy?

    by Alexey Bersenev September 6, 2014 notes

    Platelet rich plasma (PRP) is a over-hyped “regenerative medicine” product, which widely used in orthopaedics/ traumatology and sport medicine. PRP is a concentrate of platelets, isolated from patient’s blood via centrifugation (using commercially available devices). Besides authorized trials, PRP is used clinically as commercial procedure (“practice of medicine”) in point-of-care settings. What is especially interesting that it widely advertised as “cell therapy” or “stem cell therapy”. As you know, platelets are fragments of megakaryocytes, but not cells per se. And […]

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    Fetal versus maternal origin of placental mesenchymal stromal cells

    by Alexey Bersenev September 4, 2014 mesenchymal

    Mesenchymal stromal cells (MSC), isolated from human full term placenta could be fetal or maternal origin. However, the origin of ex vivo expanded placental MSC is usually not reported. The authors of recent study, attempted to analyze available published studies, in which the origin of cultured placental MSCs was assessed. MSCs cultured from human chorion have been widely assumed to be fetal in origin, despite evidence that placental MSCs may be contaminated with maternal cells, resulting in entirely maternally derived […]

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    Cells Weekly – August 31, 2014

    by Alexey Bersenev September 1, 2014 notes

    Cells Weekly is a digest of the most interesting news and events in stem cell research, cell therapy and regenerative medicine. Please give us feedback and send links to interesting news! 1. RIKEN’s interim report on STAP reproducibility RIKEN fails to reproduce STAP as per interim report, released on August 27. Histoshi Niwa is leading STAP reproducibility efforts with help of Haruko Obokata. He noted on presse-conference: “No clear signs of the STAP phenomenon could be observed so far in […]

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