Manufacturing Of Mesenchymal Stromal Cells Research Articles

Process automation in manufacturing of mesenchymal stromal cells.

Process automation in manufacturing of mesenchymal stromal cells.

Mesenchymal stromal cells: potential roles in graft‐versus‐host disease prophylaxis and treatment

Mesenchymal stromal cells: potential roles in graft‐versus‐host disease prophylaxis and treatment

Cryopreserved or Fresh Mesenchymal Stromal Cells: Only a Matter of Taste or Key to Unleash the Full Clinical Potential of MSC Therapy?

Mesenchymal stromal cells (MSCs) harbor great therapeutic potential for numerous diseases. From early clinical trials, success and failure analysis, bench-to-bedside and back-to-bench approaches, there has been a great gain in knowledge, still leaving a number of questions to be answered regarding optimal manufacturing and quality of MSCs for clinical application. For treatment of many acute indications, cryobanking may remain a prerequisite, but great uncertainty exists considering the therapeutic value of freshly thawed (thawed) and continuously cultured (fresh) MSCs. The field has seen an explosion of new literature lately, outlining the relevance of the topic. MSCs appear to have compromised immunomodulatory activity directly after thawing for clinical application. This may provide a possible explanation for failure of early clinical trials. It is not clear if and how quickly MSCs recover their full therapeutic activity, and if the "cryo stun effect" is relevant for clinical success. Here, we will share our latest insights into the relevance of these observations for clinical practice that will be discussed in the context of the published literature. We argue that the differences of fresh and thawed MSCs are limited but significant. A key issue in evaluating potency differences is the time point of analysis after thawing. To date, prospective double-blinded randomized clinical studies to evaluate potency of both products are lacking, although recent progress was made with preclinical assessment. We suggest refocusing therapeutic MSC development on potency and safety assays with close resemblance of the clinical reality.

Ex vivo expanded mesenchymal stromal cell minimal quality requirements for clinical application.

Mesenchymal stromal cells (MSCs), as advanced therapy products, must satisfy all the requirements for human use of medicinal products, aiming to maintain the quality and safety of the cells. The MSC manufacturing process for clinical use should comply with the principles of Good Manufacturing Practice (GMP). This ensures that cell preparations are produced and controlled, from the collection and manipulation of raw materials, through the processing of intermediate products, to the quality controls, storage, labeling and packaging, and release. The objective of this document is to provide the minimal quality requirements for the MSC production and its delivery for clinical use, so that the safety of the final cell therapy product will not be compromised. For this purpose, the document evaluates the most important steps of GMP-compliant MSC production: the isolation and expansion process; the validation phase of the process, including all quality controls for the characterization, functionality, potency, and safety of MSCs; and the quality control at the batch release to guarantee the safety of patient infusion.

Finessing the manufacture of mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) are an appealing cellular therapy, with early-phase clinical studies underway as therapies for acute graft-versus-host disease, inflammatory bowel disease, diabetes, wound repair and other diseases. The relative ease with which these cells can be expanded makes them especially appealing. With the use of a bone marrow aspirate, tissue culture flasks and a simple medium, MSCs can be expanded by most laboratories. One concern, however, is the type of growth factor used in the medium. The use of growth factors in cell therapy products—in particular, MSCs—is an important consideration when validating the expansion of these products for clinical use ( 1 Philippe B. Luc S. Valerie P.B. Jerome R. Alessandra B.R. Louis C. Culture and use of mesenchymal stromal cells in phase I and II clinical trials. Stem Cells Int. 2010; 2010: 503593 Crossref PubMed Scopus (53) Google Scholar , 2 Hanley P.J. Mei Z. da Graca Cabreira-Hansen M. Klis M. Li W. Zhao Y. et al. Manufacturing mesenchymal stromal cells for phase I clinical trials. Cytotherapy. 2013; 15: 416-422 Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar ). Foregoing fetal bovine serum (FBS) in favor of human platelet lysate has been reported to promote better expansion ( 3 Bieback K. Platelet lysate as replacement for fetal bovine serum in mesenchymal stromal cell cultures. Transfus Med Hemother. 2013; 40: 326-335 Crossref PubMed Scopus (142) Google Scholar ) and function of MSCs, and, unlike FBS, has not been shown to elicit immunological responses in recipients ( 4 Horwitz E.M. Gordon P.L. Koo W.K. Marx J.C. Neel M.D. McNall R.Y. et al. Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: implications for cell therapy of bone. Proc Natl Acad Sci U S A. 2002; 99: 8932-8937 Crossref PubMed Scopus (1415) Google Scholar , 5 Sundin M. Ringden O. Sundberg B. Nava S. Gotherstrom C. Le Blanc K. No alloantibodies against mesenchymal stromal cells, but presence of anti-fetal calf serum antibodies, after transplantation in allogeneic hematopoietic stem cell recipients. Haematologica. 2007; 92: 1208-1215 Crossref PubMed Scopus (248) Google Scholar ) Nevertheless, the use of platelet lysate (and FBS) is not without risk because platelet lysate has the potential to transfer adventitious agents to the recipient.

Improving immune reconstitution after cord blood transplantation using ex vivo expanded virus-specific T cells: a phase I clinical study

Ischemic stroke (IS) is the leading cause of adult disability and IS patients have few options to augment the rehabilitation process. The use of autologous mesenchymal stromal cells (MSCs) as a treatment of stroke has shown promise. One significant limitation to the use of autologousMSCs for stroke is that cells cannnot be transfused until 5 weeks post-IS due to the time required for expansion. We hypothesize that treating patients with MSCs within 48 hours of a stroke may improve outcomes. We plan to expedite the time-to-infusion by using banked allogeneic-MSCs. Here we have compared the use of an automated cell culture device (the Quantum by Terumo BCT), which uses 2.1m of hollow fibers in a bioreactor (equivalent to w120 T-175cm flasks), to our current flask-based method. In flasks, human bone marrow (BM) mononuclear cells (BMMC) were seeded at 5 105 BMMC/cm in T-175cm flasks and split 1:4 when near confluence. In the Quantum, w25 mL of unprocessed BM was added to the bioreactor. After w14 days, cells were harvested and 2.0-3.5 107 cells were re-seeded in a new bioreactor for w6 days. After 3-4 passages in flasks, an average of 2.89 108 MSCs were expanded from fourBMdonors.MSCsharvestedafter2passages in theQuantumyieldedanaverage of 6.63 108 cells. MSCs expanded in both flasks and the Quantum were analyzed by flow cytometry and met the ISCT minimum criteria for MSC (CD73, CD105,CD90 expression). Expanded MSCs were sterile, were free from chromosomal abnormalities, and differentiated into adipocytes, chondrocytes, and osteoblasts in vitro.The production of 5 108MSCs inflasks required 101-hours of labor compared to 21-hours with the Quantum. We estimated that during the manufacture of MSCs in flasks, there were 600 open events compared to 9 in the Quantum. This project is supported by NHLBI-PACT, contract # HHSN268 201000007C.

Clinical-Scale Expansion of Human Bone Marrow-Derived Mesenchymal Stromal Cells to Treat Patients After Ischemic Stroke.

Abstract 3021Ischemic stroke (IS) is the second leading cause of death worldwide and the leading cause of adult disability. IS patients have few options to limit neurological damage or augment the rehabilitation process. Tissue plasminogen activator, a fibrinolytic agent, is the only FDA approved therapy after IS but it must be administered within 3 hours of onset and is of limited benefit. Based on promising animal studies, another therapeutic option may be the application of mesenchymal stromal cells (MSCs). In one study, autologous MSCs were given to a subset of IS patients. This group showed better survival and neurological improvement, as judged by modified Rankin score and the Barthel index.A significant limitation to the use of autologous MSCs for acute stroke is that cells cannnot be transfused until at least 5 weeks after IS due to the time required for expansion in vitro. We hypothesize that treating patients with MSCs within 48 hours of a stroke may improve outcomes. We plan to expedite the time-to-infusion in two ways. First, banked allogeneic MSCs will be used instead of autologous MSCs to eliminate the production time and convert this to an off-the-shelf therapy. Second, we have compared the use of an automated cell culture device (the Quantum by Terumo BCT), which uses 2.1 m2 of hollow fibers in a bioreactor (equivalent to ∼120 T-175 cm2 flasks), to our current flask-based expansion method.In flasks, human bone marrow (BM) mononuclear cells (BMMC) were separated using the SEPAX device and were seeded at 5,000 BMMC/cm2 in T-175 cm2 flasks and split 1:4 when 70% confluent. After ∼1 month and 3–5 passages, ∼130 T-175 cm2 flasks were harvested and cryopreserved. In the Quantum, ∼25 mL of whole unprocessed BM was added to the bioreactor through a 200 μm filter. After 10 days, the cells were harvested and 2.0–3.5×107 cells were re-seeded in a new bioreactor at ∼1000 MSC/cm2 for a total culture time of ∼17 days.After 3–4 passages in flasks, an average of 2.69×108 MSCs were expanded from three independent BM donors. In two BM donors, MSCs harvested after 2 passages in the Quantum yielded an average of 8.75×108; these cells were frozen and can be used for subsequent expansions. MSCs expanded in both flasks and the Quantum were analyzed by flow cytometry and met the International Society of Cell Therapy (ISCT) minimum criteria for MSC (expression of CD73, CD105, CD90 etc). Expanded MSCs were sterile, were free from chromosomal abnormalities, and differentiated into adipocytes, chondrocytes, and osteoblasts. The production of 5×108 MSCs in flasks required 101 hours of labor compared to 21 hours with the Quantum. We estimated that during the manufacture of MSCs in flasks, there were 600 open (defined as exposing the system to the environment) events compared to 9 in the Quantum.To test the in vivo effects of manufactured MSCs, 24 aged male Long-Evans rats were randomized to receive flask-based human MSCs (P4) or saline vehicle at 7 days after stroke. At 28 days after stroke, animals treated with MSCs showed a significant reduction in neurological deficits compared with saline treated controls.In conclusion, in addition to the advantage of manufacture in a functionally- closed system, large numbers of MSCs expanded in the Quantum were available at a lower passage number with a higher average-cell yield at a slightly higher cost per 5×108 cells. When administered to rats with ischemic stroke, flask-based MSCs improved outcomes compared to placebo control treated animals. Whether MSCs expanded in the Quantum will function with equivalent efficacy is currently being tested and these data will be available for presentation in December. This project is supported by NHLBI-PACT, contract # HHSN268201000007C. Disclosures:Rice:Terumo BCT: Employment.

The therapeutic potential of dental pulp cells: more than pulp fiction?

In this issue of Cytotherapy , two papers highlight dental pulp as an important source of mesenchymal stromal cells (MSC) for potential application as cell therapy for autoimmune and related disorders. Multipotential dental pulp stem cells (DPSC) in adult human dental pulp tissue can regenerate a dentine – pulp complex in vivo and exhibit immunomodulatory properties similar to those described for bone marrow-derived MSC (BMSC) (1,2). In this issue, Demircan et al. (3) describe the immunoregulatory effects of human DPSC on T-cell subsets. They confi rm a previous report demonstrating that ex vivo -expanded DPSC suppress T-cell responses following co-culture with phytohemagglutinin-stimulated allogeneic T cells in vitro (4). Subsequent comparative studies have shown that human DPSC inhibit allogeneic mixed lymphocyte reactions and mitogen-stimulated lymphocytes when co-cultured with either BMSC or periodontal ligament stem cells (PDLSC) (2). Moreover, DPSC, BMSC and PDLSC expressed comparable gene expression levels of candidate inhibitory molecules, such as transforming growth factor (TGF)β 1, hepatocyte growth factor (HGF) and indoleamine 2,3-dioxygenase (IDO) up-regulated following coculture with activated lymphocytes or stimulation with interferon (IFN)γ . Similarly, co-cultures performed by Demircan et al. (3) showed that activated T cells stimulated DPSC to increase expression of human leukocyte antigen (HLA)-G, HGFβ 1, intracellular adhesion molecule (ICAM)-1, IDO, interleukin (IL)-6, IL-10, prostaglandin E 2 (PGE 2 ), TGFβ and vascular adhesion molecule (VCAM)-1, previously shown to mediate MSC suppression of Tcell proliferation. Furthermore, interactions between DPSC and mitogen-stimulated T cells decreased the expression of pro-infl ammatory cytokines IFNγ , IL-2, IL-6R, IL-12, IL-17A and tumor necrosis factor (TNF)α and increased expression of the anti-infl ammatory cytokine IP-10. Interestingly, CD4 CD25 Foxp3 and Treg (regulatory T-cells) cells increased when co-cultured with DPSC. This observation confi rms the ability of MSC-like populations to suppress immune and infl ammatory responses via multiple mechanisms, supporting the notion of using allogeneic DPSC preparations to treat immune-related conditions. While the immunomodulatory potential of MSClike populations derived from different tissues holds great appeal for the treatment of autoimmune and infl ammatory diseases, it must be stressed that this is not a unique property of MSC-like cells. We and others have reported that non-multipotent fi broblasts derived from various tissues exhibit comparable capacities to suppress immune responses and express many MSC-associated markers, including CD29, CD44, CD73, CD90, CD105 and CD166, while lacking expression of immune helper antigens such as HLD-DR, CD40, CD80 and CD86 (2,4,5). However, different fi broblast populations may utilize alternative mechanisms of action and could modulate varying effects on specifi c subsets of immune cells. The challenge is identifying whether specifi c MSC-like or fi broblast populations are more effi cacious for particular clinical indications using appropriate pre-clinical models, and ultimately identifying the precise mechanisms involved in these processes for each disease state. In the second paper on dental pulp cells, Bhonde et al . (6) describes the use of human platelet lysate (HPL) as a media growth supplement for the clinical scale-up of ex vivo -expanded DPSC. Most preclinical and clinical trials utilizing ex vivoexpanded (MSC)-like populations currently use fetal bovine serum (FBS). While certain batches of FBS have been approved by regulatory bodies for the manufacture of clinical-grade MSC, serum-free media may be required in the future for good manufacturing Cytotherapy, 2011; 13: 1162–1163

Translating Research into Clinical Scale Manufacturing of Mesenchymal Stromal Cells

It sounds simple to obtain sufficient numbers of cells derived from fetal or adult human tissues, isolate and/or expand the stem cells, and then transplant an appropriate number of these cells into the patient at the correct location. However, translating basic research into routine therapies is a complex multistep process which necessitates product regulation. The challenge relates to managing the expected therapeutic benefits with the potential risks and to balance the fast move to clinical trials with time-consuming cautious risk assessment. This paper will focus on the definition of mesenchymal stromal cells (MSCs), and challenges and achievements in the manufacturing process enabling their use in clinical studies. It will allude to different cellular sources, special capacities of MSCs, but also to current regulations, with a special focus on accessory material of human or animal origin, like media supplements. As cellular integrity and purity, formulation and lot release testing of the final product, validation of all procedures, and quality assurance are of utmost necessity, these topics will be addressed.

Phases I–III Clinical Trials Using Adult Stem Cells

Mesenchymal progenitor/stromal/stem cell (MSC) research has made substantial progress during the past decades. Investigators have published a wealth of basic science and preclinical data documenting the potential utility, efficacy, and safety of MSCs. The majority of this work has focused on the bone marrow- or adipose-derived MSCs. Since this body of evidence has set the stage for clinicians to advance from the bench to the bedside, Stem Cells International set out to publish a special issue devoted to the topic of Phases I–III Clinical Trials Using Adult Stem Cells. The result is a collection of ten outstanding articles submitted by investigators representing ten countries across Asia, Europe, and North America. In all cases, the methods section of each manuscript included a statement documenting that the clinical investigations were performed following institutional review board approval and/or that patient informed consent had been provided prior to the conduct of the study. Bieback et al. in Germany (Translating research into clinical scale manufacturing of mesenchymal stromal cells) and B. Philippe et al. in France (in “Culture and Use of Mesenchymal Stromal Cells in Phase I and II Clinical Trials”) have focused on the process of MSC isolation and expansion with respect to current Good Manufacturing Practices (cGMP). These authors highlight the state of the art as well as the challenges facing the development of clinical grade cells for therapeutic applications. From a regulatory perspective, the simplest application of MSC is to use them in the context of their tissue of origin. S. Akita et al. in Japan (in “Noncultured Autologous Adipose-Derived Stem Cell Therapy for Chronic Radiation Injury”) have exploited adipose-derived stem cells for soft tissue regeneration. They describe their clinical experience using autologous adipose-derived stem cells in combination with growth factors and scaffold to treat ten patients suffering from dermal radiation lesions. A series of three review papers from Belgium (“Phase 1–3 Clinical Trials Using Adult Stem Cells in Osteonecrosis and Nonunion Fractures” by J. –P. Hauzeur and V. Gangji), the USA and China (“Mesenchymal Progenitor Cells and Their Orthopedic Applications: Forging a Path towards Clinical Trials” by D. S. Shenaq et al.), and the Netherlands (“Clinical Applications of Human Mesenchymal Stromal Cells for Bone Tissue Engineering” by Chatterjea et al.) have examined the use of bone marrow-derived MSC for orthopedic applications. Each work provides a unique perspective on the published clinical trials using MSC to treat cartilage and tendon repair, critical sized defects, metabolic bone diseases, nonunion fractures, and/or osteonecrosis. From a public health perspective, MSC applications for common cardiac and central nervous system disorders could have substantial health and societal benefits. R. Sanz-Ruiz et al. in Spain (in “Phases I–III Clinical Trials Using Adult Stem Cells”) have emphasized the critical importance of randomized clinical trials to assess the utility of MSCs as they focus on the clinical evidence supporting MSC-based treatment of cardiac disease. Furthermore, they review the guidance documents on future clinical trials provided by a task force convened by the European Society for Cardiology. P. A. Walker et al. in the USA (in “Progenitor Cell Therapy for The Treatment of Central Nervous System Injury: A Review of the State of Current Clinical Trials) provide a similar perspective relating to central nervous system disorders. They evaluate the use of bone marrow-derived MSCs for ischemic stroke, spinal cord injury, and traumatic brain injury and conclude that there remains a need for further evidence to support these clinical applications. There are potential MSC applications for metabolic disorders as well. A. C. Piscaglia et al. in Italy (in “Stem Cell Therapies for Liver Disease: State of the Art and New Perspectives) have reviewed the limited number of clinical trials using hematopoietic stem cell (HSC), MSC infusion, or cytokine (G-CSF) therapy to reverse hepatic injury due to cirrhosis, drug exposure, or other causes of end stage liver disease. Likewise, A. V. Vanikar et al. from India (in “Cotransplantation of Adipose Tissue-Derived Insulin Secreting Mesenchymal Stem Cells A Novel Therapy for Insulin-Dependent Diabetes Mellitus”) describe their experience combining adipose- and bone marrow-derived MSC to treat a cohort of 11 diabetic subjects for a period of up to 23 months. They report improved hemoglobin A1C levels as well as decreased daily requirements for exogenous insulin. Uniformly, these authors highlight both the promise and the challenges faced by this emerging field of medicine. Their manuscripts identify the critical need for additional prospective, randomized controlled clinical trials evaluating all cell-based therapies, regardless of the underlying disorder. In summary, this special issue provides a snapshot of the current status of MSC-based clinical trials across the globe. Hopefully, this publication will provide a benchmark for future meta-analyses evaluating a far greater body of clinical evidence regarding the safety and efficacy of MSC therapies. Jeffrey M. Gimble Bruce A. Bunnell Louis Casteilla Jin Sup Jung Kotaro Yoshimura

Therapeutic applications of mesenchymal stromal cells

Mesenchymal stromal cells (MSC) are multipotent cells that can be derived from many different organs and tissues. They have been demonstrated to play a role in tissue repair and regeneration in both preclinical and clinical studies. They also have remarkable immunosuppressive properties. We describe their application in settings that include the cardiovascular, central nervous, gastrointestinal, renal, orthopaedic and haematopoietic systems. Manufacturing of MSC for clinical trials is also discussed. Since tissue matching between MSC donor and recipient does not appear to be required, MSC may be the first cell type able to be used as an "off-the-shelf" therapeutic product.