Characterization and Function of Cryopreserved Bone Marrow from Deceased Organ Donors: A Potential Viable Alternative Graft Source.

Despite the readily available graft sources for allogeneic hematopoietic cell transplantation (alloHCT), a significant unmet need remains in the timely provision of suitable unrelated donor grafts. This shortage is related to the rarity of certain HLA alleles in the donor pool, nonclearance of donors owing to infectious disease or general health status, and prolonged graft procurement and processing times. An alternative hematopoietic progenitor cell (HPC) graft source obtained from the vertebral bodies (VBs) of deceased organ donors could alleviate many of the obstacles associated with using grafts from healthy living donors or umbilical cord blood (UCB). Deceased organ donor-derived bone marrow (BM) can be preemptively screened, cryogenically banked for on-demand use, and made available in adequate cell doses for HCT. We have developed a good manufacturing practice (GMP)-compliant process to recover and cryogenically bank VB-derived HPCs from deceased organ donor (OD) BM. Here we present results from an analysis of HPCs from BM obtained from 250 deceased donors to identify any substantial difference in composition or quality compared with HPCs from BM aspirated from the iliac crests of healthy living donors. BM from deceased donor VBs was processed in a central GMP facility and packaged for cryopreservation in 5% DMSO/2.5% human serum albumin. BM aspirated from living donor iliac crests was obtained and used for comparison. A portion of each specimen was analyzed before and after cryopreservation by flow cytometry and colony-forming unit potential. Bone marrow chimerism potential was assessed in irradiated immunocompromised NSG mice. Analysis of variance with Bonferroni correction for multiple comparisons was used to determine how cryopreservation affects BM cells and to evaluate indicators of successful engraftment of BM cells into irradiated murine models. The t test (with 95% confidence intervals [CIs]) was used to compare cells from deceased donors and living donors. A final dataset of complete clinical and matched laboratory data from 226 cryopreserved samples was used in linear regressions to predict outcomes of BM HPC processing. When compared before and after cryopreservation, OD-derived BM HPCs were found to be stable, with CD34+ cells maintaining high viability and function after thawing. The yield from a single donor is sufficient for transplantation of an average of 1.6 patients (range, 1.2 to 7.5). CD34+ cells from OD-derived HPCs from BM productively engrafted sublethally irradiated immunocompromised mouse BM (>44% and >67% chimerism at 8 and 16 weeks, respectively). Flow cytometry and secondary transplantation confirmed that OD HPCs from BM is composed of long-term engrafting CD34+CD38-CD45RA-CD90+CD49f+ HSCs. Linear regression identified no meaningful predictive associations between selected donor-related characteristics and OD BM HPC quality or yield. Collectively, these data demonstrate that cryopreserved BM HPCs from deceased organ donors is potent and functionally equivalent to living donor BM HPCs and is a viable on-demand graft source for clinical HCT. Prospective clinical trials will soon commence in collaboration with the Center for International Blood and Marrow Research to assess the feasibility, safety, and efficacy of Ossium HPCs from BM (ClinicalTrials.gov identifier NCT05068401).

A Large-Scale Bank of Organ Donor Bone Marrow and Matched Mesenchymal Stem Cells for Promoting Immunomodulation and Transplant Tolerance.

Induction of immune tolerance for solid organ and vascular composite allografts is the Holy Grail for transplantation medicine. This would obviate the need for life-long immunosuppression which is associated with serious adverse outcomes, such as infections, cancers, and renal failure. Currently the most promising means of tolerance induction is through establishing a mixed chimeric state by transplantation of donor hematopoietic stem cells; however, with the exception of living donor renal transplantation, the mixed chimerism approach has not achieved durable immune tolerance on a large scale in preclinical or clinical trials with other solid organs or vascular composite allotransplants (VCA). Ossium Health has established a bank of cryopreserved bone marrow (BM), termed "hematopoietic progenitor cell (HPC), Marrow," recovered from deceased organ donor vertebral bodies. This new source for hematopoietic cell transplant will be a valuable resource for treating hematological malignancies as well as for inducing transplant tolerance. In addition, we have discovered and developed a large source of mesenchymal stem (stromal) cells (MSC) tightly associated with the vertebral body bone fragment byproduct of the HPC, Marrow recovery process. Thus, these vertebral bone adherent MSC (vBA-MSC) are matched to the banked BM obtained from each donor, as opposed to third-party MSC, which enhances safety and potentially efficacy. Isolation and characterization of vBA-MSC from over 30 donors has demonstrated that the cells are no different than traditional BM-MSC; however, their abundance is >1,000-fold higher than obtainable from living donor BM aspirates. Based on our own unpublished data as well as reports published by others, MSC facilitate chimerism, especially at limiting hematopoietic stem and progenitor cell (HSPC) numbers and increase safety by controlling and/or preventing graft-vs.-host-disease (GvHD). Thus, vBA-MSC have the potential to facilitate mixed chimerism, promote complementary peripheral immunomodulatory functions and increase safety of BM infusions. Both HPC, Marrow and vBA-MSC have potential use in current VCA and solid organ transplant (SOT) tolerance clinical protocols that are amenable to "delayed tolerance." Current trials with HPC, Marrow are planned with subsequent phases to include vBA-MSC for tolerance of both VCA and SOT.

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. Therefore, the availability of large single donor depots of primary MSCs would greatly benefit the cell therapy market by reducing costs associated with manufacturing. METHODS: We have discovered that an abundant population of cells possessing all the hallmarks of MSCs is tightly associated with the vertebral body (VB) bone matrix and only liberated by proteolytic digestion. Here we demonstrate that these vertebral bone-adherent (vBA) MSCs possess all the International Society of Cell and Gene Therapy-defined characteristics (e.g., plastic adherence, surface marker expression and trilineage differentiation) of MSCs, and we have therefore termed them vBA-MSCs to distinguish this population from loosely associated MSCs recovered through aspiration or rinsing of the bone marrow compartment. RESULTS: Pilot banking and expansion were performed with vBA-MSCs obtained from 3 deceased donors, and it was demonstrated that bank sizes averaging 2.9 × 108 ± 1.35 × 108 vBA-MSCs at passage 1 were obtainable from only 5 g of digested VB bone fragments. Each bank of cells demonstrated robust proliferation through a total of 9 passages, without significant reduction in population doubling times. The theoretical total cell yield from the entire amount of bone fragments (approximately 300 g) from each donor with limited expansion through 4 passages is 100 trillion (1 × 1014) vBA-MSCs, equating to over 105 doses at 10 × 106 cells/kg for an average 70-kg recipient. DISCUSSION: Thus, we have established a novel and plentiful source of MSCs that will benefit the cell therapy market by overcoming manufacturing and regulatory inefficiencies due to donor-to-donor variability.

Ischemia considerations for the development of an organ and tissue donor derived bone marrow bank.

BACKGROUND: Deceased organ donors represent an untapped source of therapeutic bone marrow (BM) that can be recovered in 3-5 times the volume of that obtained from living donors, tested for quality, cryopreserved, and banked indefinitely for future on-demand use. A challenge for a future BM banking system will be to manage the prolonged ischemia times that are inevitable when bones procured at geographically-dispersed locations are shipped to distant facilities for processing. Our objectives were to: (a) quantify, under realistic field conditions, the relationship between ischemia time and the quality of hematopoietic stem and progenitor cells (HSPCs) derived from deceased-donor BM; (b) identify ischemia-time boundaries beyond which HSPC quality is adversely affected; (c) investigate whole-body cooling as a strategy for preserving cell quality; and (d) investigate processing experience as a variable affecting quality. METHODS: Seventy-five bones from 62 donors were analyzed for CD34+ viability following their exposure to various periods of warm-ischemia time (WIT), cold-ischemia time (CIT), and body-cooling time (BCT). Regression models were developed to quantify the independent associations of WIT, CIT, and BCT, with the viability and function of recovered HSPCs. RESULTS: Results demonstrate that under "real-world" scenarios: (a) combinations of warm- and cold-ischemia times favorable to the recovery of high-quality HSPCs are achievable (e.g., CD34+ cell viabilities in the range of 80-90% were commonly observed); (b) body cooling prior to bone recovery is detrimental to cell viability (e.g., CD34+ viability < 73% with, vs. > 89% without body cooling); (c) vertebral bodies (VBs) are a superior source of HSPCs compared to ilia (IL) (e.g., %CD34+ viability > 80% when VBs were the source, vs. < 74% when IL were the source); and (d) processing experience is a critical variable affecting quality. CONCLUSIONS: Our models can be used by an emerging BM banking system to formulate ischemia-time tolerance limits and data-driven HSPC quality-acceptance standards.

The promise of organ and tissue preservation to transform medicine.

The ability to replace organs and tissues on demand could save or improve millions of lives each year globally and create public health benefits on par with curing cancer. Unmet needs for organ and tissue preservation place enormous logistical limitations on transplantation, regenerative medicine, drug discovery, and a variety of rapidly advancing areas spanning biomedicine. A growing coalition of researchers, clinicians, advocacy organizations, academic institutions, and other stakeholders has assembled to address the unmet need for preservation advances, outlining remaining challenges and identifying areas of underinvestment and untapped opportunities. Meanwhile, recent discoveries provide proofs of principle for breakthroughs in a family of research areas surrounding biopreservation. These developments indicate that a new paradigm, integrating multiple existing preservation approaches and new technologies that have flourished in the past 10 years, could transform preservation research. Capitalizing on these opportunities will require engagement across many research areas and stakeholder groups. A coordinated effort is needed to expedite preservation advances that can transform several areas of medicine and medical science.

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. 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.

Endometrial regenerative cells for treatment of heart failure: a new stem cell enters the clinic.

Heart failure is one of the key causes of morbidity and mortality world-wide. The recent findings that regeneration is possible in the heart have made stem cell therapeutics the Holy Grail of modern cardiovascular medicine. The success of cardiac regenerative therapies hinges on the combination of an effective allogeneic "off the shelf" cell product with a practical delivery system. In 2007 Medistem discovered the Endometrial Regenerative Cell (ERC), a new mesenchymal-like stem cell. Medistem and subsequently independent groups have demonstrated that ERC are superior to bone marrow mesenchymal stem cells (MSC), the most widely used stem cell source in development. ERC possess robust expansion capability (one donor can generate 20,000 patients doses), key growth factor production and high levels of angiogenic activity. ERC have been published in the peer reviewed literature to be significantly more effect at treating animal models of heart failure (Hida et al. Stem Cells 2008).Current methods of delivering stem cells into the heart suffer several limitations in addition to poor delivery efficiency. Surgical methods are highly invasive, and the classical catheter based techniques are limited by need for sophisticated cardiac mapping systems and risk of myocardial perforation. Medistem together with Dr. Amit Patel Director of Clinical Regenerative Medicine at University of Utah have developed a novel minimally invasive delivery method that has been demonstrated safe and effective for delivery of stem cells (Tuma et al. J Transl Med 2012). Medistem is evaluating the combination of ERC, together with our retrograde delivery procedure in a 60 heart failure patient, double blind, placebo controlled phase II trial. To date 17 patients have been dosed and preliminary analysis by the Data Safety Monitoring Board has allowed for trial continuation.The combined use of a novel "off the shelf" cell together with a minimally invasive 30 minute delivery method provides a potentially paradigm-shifting approach to cardiac regenerative therapy.

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. AREAS COVERED: 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. EXPERT OPINION: 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.

Exogenous endothelial cells as accelerators of hematopoietic reconstitution.

Despite the successes of recombinant hematopoietic-stimulatory factors at accelerating bone marrow reconstitution and shortening the neutropenic period post-transplantation, significant challenges remain such as cost, inability to reconstitute thrombocytic lineages, and lack of efficacy in conditions such as aplastic anemia. A possible means of accelerating hematopoietic reconstitution would be administration of cells capable of secreting hematopoietic growth factors. Advantages of this approach would include: a) ability to regulate secretion of cytokines based on biological need; b) long term, localized production of growth factors, alleviating need for systemic administration of factors that possess unintended adverse effects; and c) potential to actively repair the hematopoietic stem cell niche. Here we overview the field of hematopoietic growth factors, discuss previous experiences with mesenchymal stem cells (MSC) in accelerating hematopoiesis, and conclude by putting forth the rationale of utilizing exogenous endothelial cells as a novel cellular therapy for acceleration of hematopoietic recovery.

Expression of the epigenetic factor BORIS (CTCFL) in the human genome.

BORIS, or CTCFL, the so called Brother of the Regulator of Imprinted Sites because of the extensive homology in the central DNA binding region of the protein to the related regulator, CTCF, is expressed in early gametogenesis and in multiple cancers but not in differentiated somatic cells. Thus it is a member of the cancer testes antigen group (CTAs). Since BORIS and CTCF target common DNA binding sites, these proteins function on two levels, the first level is their regulation via the methylation context of the DNA target site and the second level is their distinct and different epigenetic associations due to differences in the non-homologous termini of the proteins. The regulation on both of these levels is extensive and complex and the sphere of influence of each of these proteins is associated with vastly different cellular signaling processes. On the level of gene expression, BORIS has three known promoters and multiple spliced mRNAs which adds another level of complexity to this intriguing regulator. BORIS expression is observed in the majority of cancer tissues and cell lines analyzed up to today. The expression profile and essential role of BORIS in cancer make this molecule very attractive target for cancer immunotherapy. This review summarizes what is known about BORIS regarding its expression, structure, and function and then presents some theoretical considerations with respect to its genome wide influence and its potential for use as a vaccine for cancer immunotherapy.

Tissue engineering, regenerative medicine, and rejuvenation in 2010: the role of adipose-derived stem cells.

Facial rejuvenation is rapidly evolving sector in the field of facial aesthetics. There is a wide variety of dermal fillers and many more are in development. Over the past few years, the study of adult-derived stem cells has become a very active area of research. Adult stem cells are an attractive option for volume restoration and facial rejuvenation. Adult stem cells are isolated from adipose tissue-adipose derived stem cells and have mesodermal, ectodermal, and endodermal potentials. Adipose-derived stem cells could conceivably be an alternative to pluripotent embryonic stem cells and could play a critical role in the rapidly expanding fields of tissue engineering and regenerative medicine. This article reviews the history of soft tissue augmentation using adipose tissue grafting and the advent of using adipose-derived stem cells. The state-of-the-art stem cell isolation technique as well as anticipated future therapeutic indications are also addressed.

Melting point equations for the ternary system water/sodium chloride/ethylene glycol revisited.

Partial phase diagrams are of considerable utility in the development of optimized cryobiological procedures. Recent theoretical predictions of the melting points of ternary solutions of interest to cryobiology have caused us to re-examine measurements that our group made for the ethylene-glycol-sodium chloride-water phase diagram. Here we revisit our previous experiments by measuring melting points at five ethylene-glycol to sodium chloride ratios (R values; R=5, 10, 15, 30, and 45) and five levels of concentration for each ratio. Melting points were averaged from three measurements and plotted as a function of total solute concentration for each R value studied. The new measurements differed from our original experimental values and agreed with predicted values from both theoretical models. Additionally, the data were fit to the polynomial described in our previous report and the resulting equation was obtained: T(m) = (38.3-2.145 x 10⁻¹ R)w + (81.19 - 2.909×10⁻¹ R)w², where w is the total solute mass fraction. This new equation provided good fits to the experimental data as well as published values and relates the determined polynomial constants to the R value of the corresponding isopleths of the three dimensional phase diagram, allowing the liquids curve for any R value to be obtained.

Container system for enabling commercial production of cryopreserved cell therapy products.

AIM: The expansion of cellular therapeutics will require large-scale manufacturing processes to expand and package cell products, which may not be feasible with current blood-banking bag technology. This study investigated the potential for freezing, storing and shipping cell therapy products using novel pharmaceutical-grade Crystal Zenith((R)) (CZ) plastic vials. MATERIALS & METHODS: CZ vials (0.5, 5 and 30 ml volume) with several closure systems were filled with mesenchymal stem cells and stored at either -85 or -196 degrees C for 6 months. Vials were tested for their ability to maintain cell viability, proliferative and differentiation capacity, as well as durability and integrity utilizing a 1-m drop test. As controls, 2 ml polypropylene vials were investigated under the same conditions. RESULTS: Post-thaw viability utilizing a dye exclusion assay was over 95% in all samples. Stored cells exhibited rapid recovery 2 h post-thaw and cultures were approximately 70% confluent within 5-7 days, consistent with nonfrozen controls and indicative of functional recovery. Doubling times were consistent over all vials. The doubling rate for cells from CZ vials were 2.14 + or - 0.83 days (1 week), 1.84 + or - 0.68 days (1 month) and 1.79 + or - 0.71 days (6 months), which were not significantly different compared with frozen and fresh controls. Cells recovered from the vials exhibited trilineage differentiation consistent with controls. As part of vial integrity via drop testing, no evidence of external damage was found on vial surfaces or on closure systems. Furthermore, the filled vials stored for 6 months were tested for container closure integrity. Vials removed from freezer conditions were transported to the test laboratory on dry ice and tested using pharmaceutical packaging tests, including dye ingress and microbial challenge. The results of all stoppered vials indicated container closure integrity with no failures. CONCLUSION: Pharmaceutical-grade plastic CZ vials, which are commercially used to package pharmaceutical products, are suitable for low-temperature storage and transport of mesenchymal stem cells, and are a scalable container system for commercial manufacturing and fill-finish operation of cell therapy products.

Combination stem cell therapy for heart failure.

Patients with congestive heart failure (CHF) that are not eligible for transplantation have limited therapeutic options. Stem cell therapy such as autologous bone marrow, mobilized peripheral blood, or purified cells thereof has been used clinically since 2001. To date over 1000 patients have received cellular therapy as part of randomized trials, with the general consensus being that a moderate but statistically significant benefit occurs. Therefore, one of the important next steps in the field is optimization. In this paper we discuss three ways to approach this issue: a) increasing stem cell migration to the heart; b) augmenting stem cell activity; and c) combining existing stem cell therapies to recapitulate a "therapeutic niche". We conclude by describing a case report of a heart failure patient treated with a combination stem cell protocol in an attempt to augment beneficial aspects of cord blood CD34 cells and mesenchymal-like stem cells.

Mesenchymal stem cells as anti-inflammatories: implications for treatment of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal X-linked musculodegenerative condition consisting of an underlying genetic defect whose manifestation is augmented by inflammatory mechanisms. Previous treatment approaches using gene replacement, exon-skipping or allogeneic cell therapy have been relatively unsuccessful. The only intervention to mediate improvement in survival, albeit minor, is glucocorticoid treatment. Given this modality appears to function via suppression of underlying inflammation; we focus this review on the inflammatory response as a target for mesenchymal stem cell (MSC) therapy. In contrast to other cell based therapies attempted in DMD, MSC have the advantages of (a) ability to fuse with and genetically complement dystrophic muscle; (b) possess anti-inflammatory activities; and (c) produce trophic factors that may augment activity of endogenous repair cells. We conclude by describing one practical scenario of stem cell therapy for DMD.

Donor-matched comparison of dental pulp stem cells and bone marrow-derived mesenchymal stem cells in a rat model.

Dental pulp stem cells (DPSCs) have drawn much interest for the regeneration of mineralized tissues, and several studies have compared DPSCs to bone marrow-derived mesenchymal stem cells (BMMSCs). However, conflicting results, possibly due to donor-associated variability, have been published and the regenerative potential of DPSCs is currently unclear. In the present study we have sought to address this problem using a donor-matched experimental design to robustly compare the biological properties of DPSCs and BMMSCs. All experiments were performed using cells isolated from a single adult Sprague-Dawley rat. Our results show that DPSCs and BMMSCs had similar morphologies and flow cytometry profiles, were capable of forming colonies in vitro and were capable of osteogenic, chondrogenic and adipogenic differentiation. However, quantitative comparisons revealed that DPSCs had a faster population doubling time and a higher percentage of stem/progenitor cells in the population, as determined by clonogenic assays. Furthermore, while both cell populations formed mineral in vitro, DPSCs had significantly higher alkaline phosphatase activity than BMMSCs after 3 weeks in osteogenic medium. These data show several key differences between DPSCs and BMMSCs and support the possibility of using DPSCs for mineralized tissue regeneration.

Optimized cryopreservation method for human dental pulp-derived stem cells and their tissues of origin for banking and clinical use.

Dental pulp is a promising source of mesenchymal stem cells with the potential for cell-mediated therapies and tissue engineering applications. We recently reported that isolation of dental pulp-derived stem cells (DPSC) is feasible for at least 120h after tooth extraction, and that cryopreservation of early passage cultured DPSC leads to high-efficiency recovery post-thaw. This study investigated additional processing and cryobiological characteristics of DPSC, ending with development of procedures for banking. First, we aimed to optimize cryopreservation of established DPSC cultures, with regards to optimizing the cryoprotective agent (CPA), the CPA concentration, the concentration of cells frozen, and storage temperatures. Secondly, we focused on determining cryopreservation characteristics of enzymatically digested tissue as a cell suspension. Lastly, we evaluated the growth, surface markers and differentiation properties of DPSC obtained from intact teeth and undigested, whole dental tissue frozen and thawed using the optimized procedures. In these experiments it was determined that Me(2)SO at a concentration between 1 and 1.5M was the ideal cryopreservative of the three studied. It was also determined that DPSC viability after cryopreservation is not limited by the concentration of cells frozen, at least up to 2x10(6) cells/mL. It was further established that DPSC can be stored at -85 degrees C or -196 degrees C for at least six months without loss of functionality. The optimal results with the least manipulation were achieved by isolating and cryopreserving the tooth pulp tissues, with digestion and culture performed post-thaw. A recovery of cells from >85% of the tissues frozen was achieved and cells isolated post-thaw from tissue processed and frozen with a serum free, defined cryopreservation medium maintained morphological and developmental competence and demonstrated MSC-hallmark trilineage differentiation under the appropriate culture conditions.

Feasibility investigation of allogeneic endometrial regenerative cells.

Endometrial Regenerative Cells (ERC) are a population of mesenchymal-like stem cells having pluripotent differentiation activity and ability to induce neoangiogenesis. In vitro and animal studies suggest ERC are immune privileged and in certain situations actively suppress ongoing immune responses. In this paper we describe the production of clinical grade ERC and initial safety experiences in 4 patients with multiple sclerosis treated intravenously and intrathecally. The case with the longest follow up, of more than one year, revealed no immunological reactions or treatment associated adverse effects. These preliminary data suggest feasibility of clinical ERC administration and support further studies with this novel stem cell type.

Electrical conductivity measurements for the ternary systems of glycerol/sodium chloride/water and ethylene glycol/sodium chloride/water and their applications in cryopreservation.

Electrical conductivity of a solution is a property that can be easily determined through the measurement of a conductivity probe. The present study demonstrates the measurements of electrical conductivity for two ternary solutions: glycerol/sodium chloride/water and ethylene glycol/sodium chloride/water. When the concentration of sodium chloride to water ratio (R) is fixed, the existence of either glycerol or ethylene glycol, both cryoprotective agents (CPAs), can be quantitatively determined by their depressive influence on electrical conductivity of the solution. The measurements were performed on solutions with a set of 10 different concentrations of CPAs, ranging from 3.2% to 50% (v/v), along with five ratios of NaCl/water solutions. Equations to fit the experimental measurements were devised to characterize the relations among electrical conductivity, CPAs concentration, and R. A conductivity meter used in this study required <5 s to read the solution's electrical conductivity, which is faster than the measurement using osmometry method. The charts of ternary solutions associated with their electrical conductivity and concentrations make it especially useful for monitoring the cryopreservation processes, including addition and removal of CPAs, to prevent osmotic damages to biological samples.

Cryopreservation and vitrification: recent advances in fertility preservation technologies.

Over the last half the 20th Century, reproductive medicine has become a critically important branch of modern medical science. Fertility preservation is a vital branch of reproductive medicine and involves the preservation of gametes (sperm and oocytes), embryos, and reproductive tissues (ovarian and testicular tissues) for use in artificial reproduction. This technology gives millions of people suffering from reproductive ailments, cancer patients who have their reproductive functions destroyed by therapy (chemotherapy and radiation) and people undergoing sterilization, a chance to conceive. The most common fertility preservation technique is cryopreservation, which involves freezing cells and tissues at cryogenic temperatures. Cryopreserved cells and tissues can endure storage for centuries with almost no change in functionality or genetic information, making this storage method highly attractive. However, developing efficient cryopreservation techniques is challenging, as both freezing and thawing exposes cells to severe stresses, potentially causing cell death. There are two major techniques for cryopreservation: freeze-thaw processes and vitrification. The major difference between them is the total avoidance of ice formation in vitrification. The use of both theoretical models that describe cell response to freezing and thawing, and experimental investigations of freezing behavior, has led to the development of successful freeze-thaw and vitrification procedures for a number of cell types. Among reproductive cells, there exist efficient cryopreservation techniques for spermatozoa and embryos. Oocytes, however, present significant hurdles in achieving successful cryopreservation, primarily due to their sensitive microtubule structure. Recently, cryopreservation of ovarian and testicular tissues has been investigated with success reported. Ovarian cryopreservation can help circumvent many of the problems associated with oocyte cryopreservation, while testicular tissue preservation may be helpful when insufficient sperm counts are available for routine semen preservation.