Identification and characterization of a large source of primary mesenchymal stem cells tightly adhered to bone surfaces of human vertebral body marrow cavities.

Background: Therapeutic allogeneic mesenchymal stromal cells (MSCs) are currently in clinical trials to evaluate their effectiveness in treating many different disease indications. Eventual commercialization for broad distribution will require further improvements in manufacturing processes to economically manufacture MSCs at scales sufficient to satisfy projected demands. A key contributor to the present high cost of goods sold for MSC manufacturing is the need to create master cell banks from multiple donors, which leads to variability in large-scale manufacturing runs.

Off the shelf cellular therapeutics: Factors to consider during cryopreservation and storage of human cells for clinical use.

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.

Manufacturing and banking of mesenchymal stem cells.

Introduction: 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.

Methylcellulose based thermally reversible hydrogel system for tissue engineering applications.

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.

Packaging Considerations for Biopreservation.

SUMMARY: The packaging system chosen for biopreservation is critical for many reasons. An ideal biopreservation container system must provide for closure integrity, sample stability and ready access to the preserved material. This means the system needs to be hermetically sealed to ensure integrity of the specimen is maintained throughout processing, storage and distribution; the system must remain stable over long periods of time as many biobanked samples may be stored indefinitely; and functionally closed access systems must be used to avoid contamination upon sample withdraw.

Preservation protocols for human adipose tissue-derived adult stem cells.

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.

Clinical grade adult stem cell banking.

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.

Cryopreservation of stromal vascular fraction of adipose tissue in a serum-free freezing medium.

Developing effective techniques for the cryopreservation of human adipose-derived adult stem cells could increase the usefulness of these cells in tissue engineering and regenerative medicine. Unfortunately, the use of serum and a commonly used cryoprotectant chemical, dimethyl sulphoxide (DMSO), during cryopreservation storage restricts the direct translation of adult stem cells to in vivo applications. The objective of this study was to test the hypothesis that the stromal vascular fraction (SVF) of adipose tissue can be effectively cryopreserved and stored in liquid nitrogen, using a freezing medium containing high molecular weight polymers, such as methylcellulose (MC) and/or polyvinylpyrollidone (PVP), as the cryoprotective agent (CPA) instead of DMSO.

Evaluation of polyvinylpyrrolidone as a cryoprotectant for adipose tissue-derived adult stem cells.

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.

Evaluation of methylcellulose and dimethyl sulfoxide as the cryoprotectants in a serum-free freezing media for cryopreservation of adipose-derived adult stem cells.

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.

Cryopreservation characteristics of adipose-derived stem cells: maintenance of differentiation potential and viability.

With the emergence of regenerative medicine, many researchers have turned to fat tissue as a source of adipose-derived stem cells (ASCs). Because freshly collected adipose tissue is not always readily available, there will be a need for improved cryopreservation methods to reproducibly maintain ASC viablility and multipotentiality in long-term storage. This study examines the efficiency of conventional dimethyl sulphoxide cryopreservation methods by measuring the maintenance of differentiation potential after one freeze cycle.

Cellular response of adipose derived passage-4 adult stem cells to freezing stress.

A differential scanning calorimeter technique was used to generate experimental data for volumetric shrinkage during cooling at 20 degrees C/min in adipose derived adult stem cells (ASCs) in the presence and absence of cryoprotective agents (CPAs). By fitting a model of water transport to the experimentally determined volumetric shrinkage data, the membrane permeability parameters of ASCs were obtained. For passage-4 (P4) ASCs, the reference hydraulic conductivity Lpg and the value of the apparent activation energy ELP were determined to be 1.

Effect of various freezing parameters on the immediate post-thaw membrane integrity of adipose tissue derived adult stem cells.

The effect of four thermal parameters on post-thaw membrane integrity of adipose tissue derived adult stem (ADAS) cells after controlled-rate freezing was investigated with the help of a two-level four-parameter (2(4)) experimental design. The four thermal parameters studied were cooling rate (CR), end temperature (ET), hold time (HT), and thawing rate (TR). Several passages, including Passage-0 (P0), Passage-1 (P1), Passage-2 (P2), Passage-3 (P3), and Passage-4 (P4), obtained from the suspended culture of stromal vascular fraction (SVF) of the ADAS cells were used for this study.

Transport phenomena during freezing of adipose tissue derived adult stem cells.

In the present study a well-established differential scanning calorimeter (DSC) technique is used to measure the water transport phenomena during freezing of stromal vascular fraction (SVF) and adipose tissue derived adult stem (ADAS) cells at different passages (Passages 0 and 2). Volumetric shrinkage during freezing of adipose derived cells was obtained at a cooling rate of 20 degrees C/min in the presence of extracellular ice and two different, commonly used, cryoprotective agents, CPAs (10% DMSO and 10% Glycerol). The adipose derived cells were modeled as spheres of 50 microm diameter with an osmotically inactive volume (Vb) of 0.

A simplified procedure to determine the optimal rate of freezing biological systems.

The effect of several cell-level parameters on the predicted optimal cooling rate B(opt) of an arbitrary biological system has been studied using a well-defined water transport model. An extensive investigation of the water transport model revealed three key cell level parameters: reference permeability of the membrane to water L(pg), apparent activation energy E(Lp), and the ratio of the available surface area for water transport to the initial volume of intracellular water (SA/WV). We defined B(opt) as the "highest" cooling rate at which a predefined percent of the initial water volume is trapped inside the cell (values ranging from 5% to 80%) at a predefined end temperature (values ranging from -5 degrees C to -40 degrees C).

A theoretically estimated optimal cooling rate for the cryopreservation of sperm cells from a live-bearing fish, the green swordtail Xiphophorus helleri.

Sperm cryopreservation of live-bearing fishes, such as those of the genus Xiphophorus is only beginning to be studied, although these fishes are valuable models for biomedical research and are commercially raised as ornamental fish for use in aquariums. To explore optimization of techniques for sperm cryopreservation of these fishes, this study measured the volumetric shrinkage response during freezing of sperm cells of Xiphophorus helleri by use of a shape-independent differential scanning calorimeter (DSC) technique. Volumetric shrinkage during freezing of X.

Cryopreservation of canine spermatozoa: theoretical prediction of optimal cooling rates in the presence and absence of cryoprotective agents.

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 degrees 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.