cell separation – Stem Cell AssaysWill Prodigy change everything?Using aptamers for cell isolationNew cell separation technologies in researchOff-the-shelf universal molecular beaconGadgets review: On-chip cell sortingOverview of cell separation technologiesNew method for cord blood processing and CD34+ cell separationClinical cell processing news – part 1, 2013Impact of density gradient centrifugation on bone marrow mononuclear cell yield and composition

http://stemcellassays.com Promoting Rigorous Reproducible Research on Stem Cells Thu, 09 Feb 2017 18:50:48 +0000 en-US hourly 1 https://wordpress.org/?v=4.4.2 http://stemcellassays.com/2016/08/will-prodigy-change-everything/ http://stemcellassays.com/2016/08/will-prodigy-change-everything/#comments Sun, 07 Aug 2016 02:45:57 +0000

Will Prodigy change everything?

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  2. Is problem of cell therapy scale up overblown?
  3. Trends in clinical use of point-of-care cell processing devices

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In the last decade advanced cell therapies are becoming highly commercialized. Industry developers, in general, favor so-called centralized model of cell product manufacturing/ delivery. In centralized model, one big manufacturing plant produces and delivers many many (thousands) products within a country or internationally. In the last few years, Biotech and cell therapy companies (Novartis, Kite Pharma, UniQure…) heavily invested in development of centralized facilities. In the other camp, academics, physicians and tools manufacturers are betting on decentralized or “point-of-care” model of cell therapy manufacturing/ delivery. You can read more about pros and cons of each model in the recent reviews – here and here. But I’d like to focus today on potential catalyst for shifting from centralized to decentralized model.

Proponents of decentralized model of cell therapy delivery would like to use relatively simple/ user-friendly, automated, closed system-based device, which can do many manufacturing processes at once. We call this type of devices “all-in-one”. Of course, “all” is not equal 100% , but combining many major processes, which are usually described in bioprocessing as separate “unit of operations”. Three years ago, German company Miltenyi Biotec has launched new device CliniMACS Prodigy (CE mark in Europe). I’d consider Prodigy’s launch as the first attempt to introduce a prototype of “all-in-one” device to clinical cell therapy world. Unlike previous generation of devices with capability to do more than one process, Prodigy went as far as integration of cell separation, magnetic cell sorting and cell culture.

Since launch, seem like Prodigy is doing well on the market (as much as I can say from attending conferences). Prodigy clinical validation reports are available now for CAR T-cells, mononuclear cells from bone marrow and peripheral blood, CD34 cell selection, NK cells and virus-specific T-cells. The main advantage of Prodigy, of course, is process integration capability. Importantly, it is fully automated, closed system device, which can save a lot on time and labor. I’d like to highlight few disadvantages of Prodigy:

  • capability to produce only one batch at time (1 patients –> 1 device)
  • high complexity and absence of backup device (equal alternative) underlies high risk
  • low flexibility of cell incubation unit (limited volume/ cell concentration)
  • relatively high cost.

Now, I’d like to discuss the main potential impact of Prodigy and devices alike. I think, Prodigy could be a catalyst for shifting from currently widely accepted centralized model to “point-of-care” model of cell manufacturing/ delivery. Devices like Prodigy is exactly what most proponents of decentralized manufacturing model were dreaming about. It could, potentially, “democratize cell therapy” by making clean room facilities obsolete and simplifying whole manufacturing process by pushing few buttons and hanging the bags. Single use disposable of multifunctional device is a replacement of class 10,000 clean rooms. Full automation is replacement of labor. Importantly, it will work nicely only for autologous (personalized) cell therapies, where cost of manufacturing is very high and starting material is very variable. In this case, cost of auto- cell product will be equal cost of manufacturing, no profit. Hospitals and patients will love it and jump on it! Allogeneic cell products manufacturing will be taken (almost entirely) by industry into centralized plants.

I think, it may work especially nicely for few processes and few indications (for example, CAR T-cells in B-ALL). If your hospital wants to have multiple cell therapies for multiple indications, Prodigy may not be universal answer. Ideally, big multidisciplinary hospital would have cell therapy facility with both types devices “all-in-one” and “plug-and-play” (multiple devices for multiple processes). But if you’re looking only for getting your CAR T-cell program going in hem/oncology on a budget, Prodigy alone could be the solution.

Finally, I’d like to notice that Prodigy is just at the beginning of its way in clinical cell therapy. We may see more and better “all-in-one” devices in the future. We don’t know for sure if fundamental shift from centralized to hospital-based model will finally happen for some autologous cell products in some indications. Miltenyi’s people firmly believe (based on what I’ve learned from few talks on different conferences) that it may take years, but finally decentralized “point-of-care” model will prevail for autologous cell therapies.

What do you think? Will Prodigy change everything?

Disclaimer: This post is neither advertisement nor endorsement, but invitation for discussion. I’m not a user of CliniMACS Prodigy. I do not have any conflicts, related to Miltenyi Biotec.

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Using aptamers for cell isolation

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  1. Mesenchymal stem cells isolation in one step
  2. Feasibility of magnetic isolation CD133+ cells from human cord blood
  3. Cell tracking techniques in vivo

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Aptamers are single-stranded RNA or DNA oligonucleotides, which could be screened and synthesized for specific target (including any cell type), using systematic evolution of ligands by exponential enrichment (SELEX) technology. Aptamers are known for more than two decades and have been used successfully for molecular diagnostics, targeted therapy, biomarkers discovery, as biosensensors for cell labeling and other applications. Cell-SELEX technology was used for aptamer-based isolation of cells in cancer and stem cell research (reviewed here). In cell therapy, aptamers could be used for (1) cell tracking (reviewed here) and (2) cell isolation. I’d like to focus on application of aptamers for cell isolation.

In research, aptamers have been used for isolation of mesenchymal stromal cells, embryonic stem cells and immune cells. However, to my knowledge, there are no reports on use of aptamers in cell therapy clinical trials (for cell tracking or cell isolation purposes). I was able to find only one research paper, which experimentally assesses therapeutic value of aptamer-isolated cells.

Aptamer-based cell isolation is very similar to antibodies. Compared to antibodies, multiple authors are indicating the following advantages of aptamers:

  • small size allows wider bioavailability
  • lack of immunogenicity
  • easy chemical synthesis and scale-out manufacturing
  • easy to modify
  • high stability
  • possibility of decoupling from the cells.

Aptamers and antibodies have comparable specificity and result in similar cell yield after isolation. Potential disadvantages of aptamers in cell therapy are the following:

  • few clones should be tested for specificity
  • non-specific binding with other types of cells, optimization required
  • relatively low cell yield after decoupling
  • not qualified (approved) for clinical cell isolation
  • unknown safety (not rigorously tested for biocompatibility).

Study from S. Korea, published last year in PLoS ONE, is the most rigorous (as of today) pre-clinical assessment of aptamers for cell isolation. I’d highly recommend to read this study if you’re interested in cell isolation techniques.

The concept of using aptamers in clinical cell manufacturing looks very attractive to me. I’m not sure why this approach is not gaining momentum in cell therapy. If you ever have worked with aptamers and read on this topic, please share your thoughts on its potential application in cell therapy. Also, if you know any manufacturer of clinical-grade aptamers, please let me know.

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New cell separation technologies in research

Related posts:

  1. Overview of cell separation technologies
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  3. Magnetic cell separation in plain english

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The current research market of cell separation is hugely dominated by three companies – Stem Cell Technologies (Canada), Miltenyi Biotec (Germany) and Thermo Fisher (Dynal brand). Everyone, who involved in cell research, knows these brands and use their products. After 2 decades, magnetic cell separation methods, developed by these companies, became “conventional” or standard in research. Today, I’d like to look beyond “conventional” brands and briefly overview new comers to the world of cell separation. New companies with new technologies are diversifying the market and offering some advantages. I’d like crowdsource here – please feel free to add new companies if I missed them.

PluriSelect
German company, which offers non-magnetic cell isolation kits and reagents for researchers. Competitive advantage of their technology is simultaneous and/ or sequential isolation of multiple cell populations.
Learn more from video tutorials.
Also, check this protocol: Cascade System for Simultaneous Separation of Multiple Cell Types

Quad Technologies
US-based startup, which entered cell separation market last year. Company offers hydrogel-based technology for magnetic tag-free cell separation. Competitive advantages, listed by company.

Akadeum
US-based company, founded in 2012. Akadeum offers new non-magnetic technology for cell separation – buoyancy-activated cell sorting (BACS). Buoyancy technology entails microscopic microbubbles, which float on a surface of the liquid after capturing targeted cells. Competitive advantages – simplicity, speed and magnet/ device independence.

BioMagnetic Solutions
US-based company, which offers improved magnetic cell isolation. Company makes ferrofliuds (nano-magnetic liquids) and magnetic devices. Competitive advantage – increased efficiency and purity due to special qualities of ferrofluids.

CellectCell
Spinout of Georgia Tech, utilizing microfluidic-based technology. Company offers label-free isolation of adherent cells in microfluidic chamber. Competitive advantages – label-free, speed (95% purity in 5 min) and focus on adherent cells.

BioCEP
Israeli company, founded in 2006. BioCEP offers improved automated magnetic cell separation. Competitive advantage is highly pure isolation of rare cell populations.

If you had a chance to try cell separation systems, mentioned in this post, please share your experience!

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Off-the-shelf universal molecular beacon

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Molecular beacon (MB) is a new tool to isolate cells, based on intracellular genetic marker. Specific molecular beacons were designed for positive selection of particular cell types. The next step in development of MB – universal RNA beacon – was recently described:

Here, we report on an off-the-shelf universal beacon that targets a nonsense tag placed in the untranslated region of a functional protein or even a biomarker. We show that UB technology allows for detection and high-throughput separation of any exogenous gene without altering the properties of the protein product.

The beacon was designed for detection of exogenously introduced genes. The study describes universal MB design. Beacon has a fluorescent tag for sorting by FACS. The authors tested it in positive and negative selection for 2 markers – pluripotency gene Nanog and pacemaker channel gene HCN2. They were able to select Nanog+ cells with purity ~99%.

The authors highlight advantages of universal beacon:

… developing a successful, specific beacon to a particular transfected gene can take months to develop and in some cases is impossible. Here, we report on an off-the-shelf universal beacon that decreases the time and cost of applying beacon technology to select any living cell population transfected with an exogenous gene.

I found this approach very interesting and promising. As of now – for research. What do you think?

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Gadgets review: On-chip cell sorting

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  3. Fluorescence-Activated Cell Sorting for therapeutic cell products

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Development of microfluidic chips allows to overcome some disadvantages of conventional cell sorting methodologies – FACS and MACS. Sort-on-a-chip is getting closer to the clinic! In this brief overview, I’ll highlight advantages of on-chip cell sorting and introduce few players on a market.

What are the advantages of on-chip cell sorting in comparison to FACS/ MACS?

  • no aerosol
  • higher viability (less cell damage via reduction of shear stress)
  • single use disposables
  • no contamination issues
  • easy to use
  • portable (compact) devices
  • potential for GMP-compliance
  • potential for closed system
  • possibility of label-free sorting

Most on-chip sorting systems are currently made for research. The ultimate goal, of course, is to bring on-chip cell sorting to the clinic. On this end, more work should be done to close system completely, address questions of sorting speed, scalability and cross-contamination. Let’s look what we have today on the market:

On-chip cell sorting available systems overview

Nanocellect Biomedical
Technology utilizes “a low voltage piezoelectric acuator“. Flow cytometer/ sorter – WOLF cell sorter (coming soon). For research use only.
Nanocellect promo video:

Sony Biotechnology
Sony Cell Sorter SH800 utilizes “Blu-ray Disc technology”. For research use only. Read more about chip and technology.

On-chip Biotechnologies
Technology: Chips with two sheath flow and “sorting by pulse flows”. Company sells analyzer, sorter and chips. For research use only.
On-chip Biotechnologies promo video:

Owl Biomedical (now part of Miltenyi Biotec)
Owl Biomedical was the first company, which made decision to bring on-chip sort to the clinic. The company developed closed system, high speed, scalable, GMP-compliant on-chip sorting. Technology based on micro electro mechanical systems (MEMS). Product: Nanosorter. The company was acquired by Miltenyi Biotec last year. Currently under clinical development.
Watch Owl Biomedical presentation:

To conclude: On-chip cell sorting field is developing very rapidly. Using disposable chips allow to overcome many disadvantages of conventional FACS technology. Cell sort on chip is currently moving from research applications to clinical use. Many technologies, which I didn’t mention in this review still under development in academic labs. Market of on-chip cell sorting here to stay and to grow!

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PS: Special thanks to Eric Rentschler and Jun Seita.

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http://stemcellassays.com/2014/03/on-chip-cell-sorting/feed/ 1 http://stemcellassays.com/2013/03/overview-cell-separation-tehcnologies/ http://stemcellassays.com/2013/03/overview-cell-separation-tehcnologies/#respond Sun, 24 Mar 2013 01:32:37 +0000

Overview of cell separation technologies

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The recent review Stem cell separation technologies, comprehensively covers all current cell separation methods. The review includes classification of cell separation, overview of methods, pros and cons, challenges and future directions.

Cell separation techniques can be broadly classified into two categories: techniques based on physical parameters (size/density), and techniques based on affinity (chemical, electrical, or magnetic couplings).

The authors cover such separation techniques as:

  • fluorescence-activated cell sorting (FACS);
  • magnet-activated cell sorting (MACS);
  • preplating;
  • conditioned expansion media;
  • density gradient centrifugation;
  • field flow fractionation (FFF);
  • dielectrophoresis (DEP);
  • aqueous two-phase system;
  • systematic evolution of ligands by exponential enrichment (SELEX);
  • microfluidic platforms.

On future development:

Among the many stem cell separation methods, affinity-based approaches are so far most efficient and reliable, due to the high specificity of antibodies that recognize stem cell surface markers. FACS can achieve an impressive >95% purity, while MACS is portable. With growing knowledge on better stem cell markers, and the generation of more specific aptamers by SELEX, affinity-based techniques will still be very powerful in the future for stem cell separation.

As a method to isolate stem cells in an automated, miniaturized, multiplex, and portable fashion, microfluidics offer exciting solutions to these challenges. Although the purity of stem cell separation by microfluidic devices needs to be substantially improved, merging conventional technologies onto microfluidic platform will be extremely beneficial.

Most separation methods have room to increase throughput. Among them, FFF, DEP, and density gradient centrifugation show great potential in large-scale production.

This is the best review that I ever read on this subject. Highly recommended!

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New method for cord blood processing and CD34+ cell separation

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Hematologist Jay Mehrishi from U of Cambridge published a very interesting review, where he criticizes the current methods of cord blood processing and CD34+ cell separation methodology. This is very interesting and unique review and absolutely worth a look!

He discusses the use of different reagents and temperature during typical cord blood processing and highlights the flaws. His review focuses on such methodological issues as use of appropriate temperature, HESPAN (red blood cell sedimentation agent), DNase and other enzymes. He went through possible issues with magnetic positive selection of CD34+ cells by commercially available Miltenyi Biotech reagents and CliniMACS procedure.

Miltenyi particles internalized by cells could release iron that accumulating in liver or spleen would then risk toxicity.
… Omitting the positive selection with antibody-linked Miltenyi particles obviates the use of harsh reagents to release the cells. Internalized Miltenyi particles are a toxicity hazard that needs investigations.

Mehrishi think that the current methodologies, widely used for cord blood processing, lead to great loss of CD34+ cells, damage cells and could compromise their function. He noted, that most of current methods allow to enrich CD34+ cells only as much as 0.13 – 0.3% of total mononuclear cells.

In the following original study, Mehrishi with a co-author propose a novel methodology for cord blood processing and CD34+ cell enrichment. They used so-called physicochemical charge-based methods, including (1) nylon wool column (NWC), (2) direct rosetting with sheep red blood cells (SRBCs) and (3) avoiding incubation in the cold. Importantly, new methodology allows to avoid magnetic (Miltenyi) particles and HESPAN.

The authors were able to get much better recovery of CD34+ cells than by current (positive selection) methods:

CD34+ cell yields (approx. 5.12%) were 39 times greater than 0.13% (Korean study, 11,098 UCB units) and 10 to 20 times greater than 0.25% to 0.3% harvested by anti-CD34 Miltenyi particles.

Interestingly, SRBCs associated with significant reduction of T-cells:

The physicochemical approach, specifically the direct SRBC rosetting that ensures retaining immature preprogenitors, reduced the number of mature CD3+ T cells below 10% (Table 2, bottom row, Experiments 2, 3, and 5, Columns 5, 6, and 7). This is important because one of the gravest hazards and risks in transplantation arises from mature T cells responsible for GVHD.

In the discussion, the authors also noted:

The procedure described herein is manageable in a closed-bag system under GMP conditions, such as already developed.

In conclusion:

Achieving approximately 5% yields of CD34+ cells (153 × 105/110 mL cord-placenta blood) is a major advance holding great promise, for the first time increasing the prospect of stem cell therapy of 70-kg adults, using a single UCB donation (with dose of 1.5 × 105 cells/kg) and considerably cheaper cultured red blood cells manufacture (multiple packs/2 × 1012).

I think, these data are impressive and very interesting. I wonder if rosetting with sheep red blood cells was ever cleared as clinically grade (for human use)? What do you think folks? I’d value your opinions!

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Clinical cell processing news – part 1, 2013

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  3. Clinical cell processing news – part 1

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Clinical Cell Processing News series highlights and reviews new products and techniques for clinical-grade cell processing and manufacturing. Cell processing devices, cultureware, bioreactors, GMP-grade reagents, cell separation techniques… If you would like to promote your product or platform, contact us!

1. Validation of a novel marrow filter device (Kaneka) for mesenchymal stromal cells enrichment (Cytotherapy)

Human bone marrow cells from eight healthy donors were processed using a marrow filter device and, in parallel, using buoyant density centrifugation by two independent investigators.
The marrow filter device generated significantly greater initial cell recovery requiring less investigator time and resulted in approximately 2.5-fold more MSCs after the second passage. The immunophenotype and differentiation potential of MSCs isolated using the two methods were equivalent and consistent with the defining criteria.

2. Short-term stability of human expanded mesenchymal stem cells after harvest (Cytotherapy)

We examined the viability, self-renewal capacity and differentiation capability of MSCs on short-term in vitro storage in saline or dextrose solution at 4°C and room temperature.
Storage of culture-harvested MSCs for >2 h is likely to result in suboptimal MSC-mediated tissue regeneration because of decreased cell viability and differentiation capacity.

3. Impact of storage temperature and processing delays on cord blood quality (Transfusion)

We compared units stored at room temperature (RT) or at 4°C for 72 hours before cryopreservation to units processed shortly after collection (

4. Pre-clinical evaluation of CellCap device for harvesting and selecting adipose stem cells (PLoS ONE)

This study utilised primary rat adipose to validate a novel strategy for selecting adult stem cells. Experiments explored the use of large, very dense cell-specific antibody loaded isolation beads (diameter 5x–10x greater than target cells) which overcome the problem of endocytosis and have proved to be very effective in cell isolation from minimally processed primary tissue.

5. Stability of cell populations in cord blood stored 96 hours at room temperature before processing (Transfusion)

CD34+ cells and mature T lymphocytes increased (viability 99%). Mature B lymphocytes and MSCs decreased, maintaining viability. Granulocytes decreased with loss of viability. Monocytes and immature B lymphocytes remained stable. Clonogenic assays showed a decrease in colony-forming unit (CFU) number in UCB units stored for 96 hours.

UCB manipulation did not influence cell viability. All cell subsets remained viable until 96 hours after collection.

6. Pre-clinical evaluation of Tisseel fibrin spray system for delivery of adipose-derived stromal vascular fraction (Cytotherapy)

Our results indicate that mesenchymal and endothelial progenitor cells, prepared in a closed system from unpassaged lipoaspirate samples, retain their growth and differentiation capacity when applied and immobilized on a substrate using a clinically approved fibrin sealant spray system.

7. Development computerized system for clinical cell processing, collection and administration (J Oncol Pract)

… Partners Healthcare System Information Services (PHS-IS; Boston, MA) has worked with oncologists and staff in the cell processing laboratory at the Dana-Farber Cancer Institute (Boston, MA) to develop and implement a novel, comprehensive computerized system for physician ordering and management of cellular products. A multidisciplinary team was formed to accomplish the task of developing a cellular product management system.
In addition, the biotherapy applications were put into use by additional groups of providers when the Dendreon (Seattle, WA) product sipuleucel-T received FDA approval for treatment of patients with prostate cancer.

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Impact of density gradient centrifugation on bone marrow mononuclear cell yield and composition

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