Mesenchymal stem cells (MSC) and MSC-like cells hold great promise and offer many advantages for developing effective cellular therapeutics. Current trends indicate that the clinical application of ...MSC will continue to increase markedly. For clinical applications, large numbers of MSC are usually required, ideally in an off-the-shelf format, thus requiring extensive MSC expansion ex vivo and subsequent cryopreservation and banking.
To exploit the full potential of MSC for cell-based therapies requires overcoming significant cell-manufacturing, banking and regulatory challenges. The current review will focus on the identification of optimal cell source for MSC, the techniques for production scale-up, cryopreservation and banking and the regulatory challenges involved.
There has been considerable success manufacturing and cryopreserving MSC at laboratory scale. Surprisingly little attention, however, has been given to translate these technologies to an industrial scale. The development of cost-effective advanced technologies for producing and cryopreserving commercial-scale MSC is important for successful clinical cell therapy.
Abstract The field of cellular therapeutics has immense potential, affording an exciting array of applications in unmet medical needs. One of several key issues is an emphasis on getting these ...therapies from bench to bedside without compromising safety and efficacy. The successful commercialization of cellular therapeutics will require many to extend the shelf-life of these therapies beyond shipping “fresh” at ambient or chilled temperatures for “just in time” infusion. Cryopreservation is an attractive option and offers potential advantages, such as storing and retaining patient samples in case of a relapse, banking large quantities of allogeneic cells for broader distribution and use and retaining testing samples for leukocyte antigen typing and matching. However, cryopreservation is only useful if cells can be reanimated to physiological life with negligible loss of viability and functionality. Also critical is the logistics of storing, processing and transporting cells in clinically appropriate packaging systems and storage devices consistent with quality and regulatory standards. Rationalized approaches to develop commercial-scale cell therapies require an efficient cryopreservation system that provides the ability to inventory standardized products with maximized shelf life for later on-demand distribution and use, as well as a method that is scientifically sound and optimized for the cell of interest. The objective of this review is to bridge this gap between the basic science of cryobiology and its application in this context by identifying several key aspects of cryopreservation science in a format that may be easily integrated into mainstream cell therapy manufacture.
Developing effective techniques for the cryopreservation of human adipose-derived adult stem cells (ASCs) could increase the usefulness of these cells in tissue engineering and regenerative medicine. ...To this end, we investigated the post-freeze/thaw viability and apoptotic behavior of Passage 1 (P1) adult stem cells (ASCs) in 11 different media: (i) the traditional media containing Dulbecco's modified Eagle's medium (DMEM) with 80% fetal calf serum (FCS) and 10% dimethyl sulfoxide (DMSO), (ii) DMEM with 80% human serum (HS) and 10% DMSO, (iii) DMEM with 1% methyl cellulose (MC) and 10% of either HS or FCS or DMSO, and (iv) DMEM with 0%, 2%, 4%, 6%, 8%, or 10% DMSO. Approximately 1 mL (10(6) cells/mL) of P1 ASCs were frozen overnight in a -80 degrees C freezer and stored in liquid nitrogen for 2 weeks before being rapidly thawed in a 37 degrees C water bath (1-2 min of agitation), resuspended in culture media, and seeded in separate wells of a 6-well plate for a 24-h incubation period at 37 degrees C. After 24 h, the thawed samples were analyzed by bright-field microscopy and flow cytometry. The results suggest that the absence of DMSO (and the presence of MC) significantly increases the fraction of apoptotic and/or necrotic ASCs. However, the percentage of viable cells obtained with 2% DMSO and DMEM was comparable with that obtained in freezing media with 10% DMSO and 80% serum (HS or FCS), that is, approximately 84% +/- 5% and approximately 84% +/- 8%, respectively. Adipogenic and osteogenic differentiation behavior of the frozen thawed cells was also assessed using histochemical staining. Our results suggest that post-thaw ASC viability, adipogenic and osteogenic differentiability can be maintained even when they are frozen in the absence of serum but with a minimal concentration of 2% DMSO in DMEM.
The thermoresponsive behavior of a Methylcellulose (MC) polymer was systematically investigated to determine its usability in constructing MC based hydrogel systems in cell sheet engineering ...applications. Solution-gel analyses were made to study the effects of polymer concentration, molecular weight and dissolved salts on the gelation of three commercially available MCs using differential scanning calorimeter and rheology. For investigation of the hydrogel stability and fluid uptake capacity, swelling and degradation experiments were performed with the hydrogel system exposed to cell culture solutions at incubation temperature for several days. From these experiments, the optimal composition of MC-water-salt that was able to produce stable hydrogels at or above 32 °C, was found to be 12% to 16% of MC (Mol. wt. of 15,000) in water with 0.5× PBS (~150mOsm). This stable hydrogel system was then evaluated for a week for its efficacy to support the adhesion and growth of specific cells in culture; in our case the stromal/stem cells derived from human adipose tissue derived stem cells (ASCs). The results indicated that the addition (evenly spread) of ~200 µL of 2 mg/mL bovine collagen type -I (pH adjusted to 7.5) over the MC hydrogel surface at 37 °C is required to improve the ASC adhesion and proliferation. Upon confluence, a continuous monolayer ASC sheet was formed on the surface of the hydrogel system and an intact cell sheet with preserved cell-cell and cell-extracellular matrix was spontaneously and gradually detached when the grown cell sheet was removed from the incubator and exposed to room temperature (~30 °C) within minutes.
Clinical grade adult stem cell banking Thirumala, Sreedhar; Goebel, W. Scott; Woods, Erik J.
Organogenesis,
07/2009, Letnik:
5, Številka:
3
Journal Article
Recenzirano
Odprti dostop
There has been a great deal of scientific interest recently generated by the potential therapeutic applications of adult stem cells in human care but there are several challenges regarding quality ...and safety in clinical applications and a number of these challenges relate to the processing and banking of these cells ex-vivo. As the number of clinical trials and the variety of adult cells used in regenerative therapy increases, safety remains a primary concern. This has inspired many nations to formulate guidelines and standards for the quality of stem cell collection, processing, testing, banking, packaging and distribution. Clinically applicable cryopreservation and banking of adult stem cells offers unique opportunities to advance the potential uses and widespread implementation of these cells in clinical applications. Most current cryopreservation protocols include animal serum proteins and potentially toxic cryoprotectant additives (CPAs) which prevent direct use of these cells in human therapeutic applications. Long term cryopreservation of adult stem cells under good manufacturing conditions using animal product free solutions is critical to the widespread clinical implementation of ex-vivo adult stem cell therapies. Furthermore, to avoid any potential cryoprotectant related complications, reduced CPA concentrations and efficient post-thaw washing to remove CPA are also desirable. The present review focuses on the current strategies and important aspects of adult stem cell banking for clinical applications. These include current good manufacturing practices (cGMPs), animal protein free freezing solutions, cryoprotectants, freezing & thawing protocols, viability assays, packaging and distribution. The importance and benefits of banking clinical grade adult stem cells are also discussed.
The objective of this study was to test the hypothesis that human adipose tissue-derived adult stem cells (ASCs) can be effectively cryopreserved and stored in liquid nitrogen using a freezing medium ...containing a high-molecular-weight polymer, polyvinylpyrrolidone (PVP), as the cryoprotective agent (CPA) instead of dimethylsulfoxide (DMSO). To this end we investigated the postfreeze/thaw viability and apoptotic behavior of passage 1 ASCs cryopreserved in 15 different media: (i) the traditional media containing Dulbecco's modified Eagle's medium (DMEM) with 80% fetal calf serum (FCS) and 10% DMSO; (ii) DMEM with 80% human serum (HS) and 10% DMSO; (iii) DMEM with various concentrations (1%, 5%, 10%, 20%, and 40%) of PVP as the sole CPA; (iv) DMEM with PVP (5%, 10%, and 20%) and HS (10%); (v) DMEM with PVP (5%, 10%, and 20%) and FCS (10%); and (vi) DMEM with PVP (10%) and FCS (40% and 80%). Approximately 1 mL (10(6) cells/mL) of passage 1 ASCs were frozen overnight in a -80 degrees C freezer and stored in liquid nitrogen for 2 weeks before being rapidly thawed in a 37 degrees C water bath (1-2 min of agitation), resuspended in culture media, and seeded in separate wells of a six-well plate for a 24-h incubation period at 37 degrees C. After 24 h, the thawed samples were analyzed by bright-field microscopy and flow cytometry. The results suggest that the absence of DMSO significantly increases the fraction of apoptotic and/or necrotic ASCs. However, the percentage of viable cells obtained with 10% PVP and DMEM was comparable with that obtained in freezing media with DMSO and serum (HS or FCS), that is, approximately 70% + or - 8% and approximately 83% + or - 8%, respectively. Slightly enhanced cell viability was observed with the addition of serum (either HS or FCS) to the freezing media containing PVP as the CPA. Adipogenic and osteogenic differentiation behaviors of the frozen thawed cells were also assessed using histochemical staining and optical density measurements and the expression of adipogenic-associated genes was analyzed using reverse transcription-polymerase chain reaction. Our results suggest that after thawing, ASC viability and adipogenic and osteogenic differentiation abilities can be maintained even when ASCs are frozen in the absence of serum but with 10% PVP in DMEM.
The development of simple but effective storage protocols for adult stem cells will greatly enhance their use and utility in tissue-engineering applications. There are three primary storage ...techniques, freezing (cryopreservation), drying (anhydrobiosis), and freeze drying (lyophilization), each with its own advantages and disadvantages. Cryopreservation has shown the most promise but is a fairly complex process, necessitating the use of chemicals called cryoprotective agents (CPAs), freezing equipment, and obviously, storage in liquid nitrogen. Preservation by desiccation is an alternative that attempts to reproduce a naturally occurring preservative technique, namely, the phenomenon of anhydrobiosis and requires the use of high (and possibly, toxic) concentration of CPAs as well as disaccharides (sugars). Lyophilization works by first cryopreserving (freezing) the material and then desiccating (drying) it by the process of sublimation or the conversion of ice (solid) to water vapor (gas phase). The purpose of this chapter is to present a general overview of these storage techniques and the optimal protocols/results obtained in our laboratory for long-term storage of adult stem cells using freezing storage and drying storage.
In the present study a shape independent differential scanning calorimeter (DSC) technique was used to measure the dehydration response during freezing of ejaculated canine sperm cells. Volumetric ...shrinkage during freezing of canine sperm cell suspensions was obtained at cooling rates of 5 and 10
°C/min in the presence of extracellular ice and CPAs (6 different combinations of freezing media were used, ranging from a media with no CPAs, and those with 0.5%, 3%, and 6% glycerol and with 0.5% and 3% Me
2SO). Using previously published data, the canine sperm cell was modeled as a cylinder of length 105.7
μm and a radius of 0.32
μm with an osmotically inactive cell volume,
V
b, of 0.6
V
o, where
V
o is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data the best fit membrane permeability parameters (
L
pg and
E
Lp) were determined. The “combined best fit” membrane permeability parameters at 5 and 10
°C/min for canine sperm cells in the absence of CPAs are:
L
pg=0.52×10
−15
m
3/Ns (0.0029
μm/min-atm) and
E
Lp=64.0
kJ/mol (15.3
kcal/mol) (
R
2=0.99); and the corresponding parameters in the presence of CPAs ranged from
L
pgcpa=0.46 to 0.53
×
10
−15 m
3/Ns (0.0027–0.0031
μm/min-atm) and
E
Lpcpa=46.4–56.0
kJ/mol (11.1–13.4
kcal/mol). These parameters are significantly different than previously published parameters for canine and other mammalian sperm obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice. The parameters obtained in this study also suggest that optimal rates of freezing canine sperm cells ranges from 10 to 30
°C/min; these theoretical cooling rates are found to be in close conformity with previously published but empirically determined optimal cooling rates.
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