Long-term Cryopreserved Research Articles

Quality Assessment of Long-Term Cryopreserved Human Bone-Derived Marrow Mesenchymal Stromal Cell Samples: Experience from the Texas Heart Institute Biorepository and Biospecimen Profiling Core.

In biomedical research, biorepositories are pivotal resources that safeguard and supply clinical samples for scientific investigators. Proper long-term cryopreservation conditions are essential to maintain biospecimen quality. In this study, we analyzed the efficacy of sample cryopreservation at the Texas Heart Institute Biorepository and Biospecimen Profiling Core (THI-BRC). Our assessments included a thorough review of internal processes, quality reports, and both internal and external audit outcomes. We examined the integrity of human bone marrow-derived multipotent mesenchymal stromal cells (BM-MSCs) that were cryopreserved for over 5 years. These samples originated from randomly selected clinical trial participants or commercially sourced cell lines. Parameters such as cell viability, DNA and RNA integrity, population doubling time, sterility, and BM-MSC-specific attributes such as surface antigen expression and differentiation potential were studied. BM-MSC samples cryopreserved for ∼6 months served as our control. Our results demonstrated that the 5-year cryopreserved samples maintained their integrity compared with the shorter-term stored control samples. Moreover, THI-BRC has met accreditation agency standards and has not received any repeated deficiencies over 7 years. Collectively, our findings affirm that THI-BRC's biospecimen storage protocols align with accepted standards as confirmed by the quality assessment of long-term stored clinical samples.

Fetal Kidney Grafts and Organoids from Microminiature Pigs: Establishing a Protocol for Production and Long-Term Cryopreservation.

Fetal organs and organoids are important tools for studying organ development. Recently, porcine organs have garnered attention as potential organs for xenotransplantation because of their high degree of similarity to human organs. However, to meet the prompt demand for porcine fetal organs by patients and researchers, effective methods for producing, retrieving, and cryopreserving pig fetuses are indispensable. Therefore, in this study, to collect fetuses for kidney extraction, we employed cesarean sections to preserve the survival and fertility of the mother pig and a method for storing fetal kidneys by long-term cryopreservation. Subsequently, we evaluated the utility of these two methods. We confirmed that the kidneys of pig fetuses retrieved by cesarean section that were cryopreserved for an extended period could resume renal growth when grafted into mice and were capable of forming renal organoids. These results demonstrate the usefulness of long-term cryopreserved fetal pig organs and strongly suggest the effectiveness of our comprehensive system of pig fetus retrieval and fetal organ preservation, thereby highlighting its potential as an accelerator of xenotransplantation research and clinical innovation.

Quantification of Analgesic and Anti-Inflammatory Lipid Mediators in Long-Term Cryopreserved and Freeze-Dried Preserved Human Amniotic Membrane.

The aim of this study was to compare concentrations of endogenous N-acylethanolamine (NAE) lipid mediators-palmitoylethanolamide (PEA), oleoylethanolamide (OEA), and anandamide (AEA)-in fresh, decontaminated, cryopreserved, and freeze-dried amniotic membrane (AM) allografts, thereby determining whether AM's analgesic and anti-inflammatory efficiency related to NAEs persists during storage. The concentrations of NAEs were measured using ultra-high-performance liquid chromatography-tandem mass spectrometry. Indirect fluorescent immunohistochemistry was used to detect the PEA PPAR-α receptor. The concentrations of PEA, OEA, and AEA were significantly higher after decontamination. A significant decrease was found in cryopreserved AM compared to decontaminated tissue for PEA but not for OEA and AEA. However, significantly higher values for all NAEs were detected in cryopreserved samples compared to fresh tissue before decontamination. The freeze-dried AM had similar values to decontaminated AM with no statistically significant difference. The nuclear staining of the PPAR-α receptor was clearly visible in all specimens. The stability of NAEs in AM after cryopreservation was demonstrated under tissue bank storage conditions. However, a significant decrease, but still higher concentration of PEA compared to fresh not decontaminated tissue, was found in cryopreserved, but not freeze-dried, AM. Results indicate that NAEs persist during storage in levels sufficient for the analgesic and anti-inflammatory effects. This means that cryopreserved AM allografts released for transplant purposes before the expected expiration (usually 3-5 years) will still show a strong analgesic effect. The same situation was confirmed for AM lyophilized after one year of storage. This work thus contributed to the clarification of the analgesic effect of NAEs in AM allografts.

Long-Term Cryopreservation of Nasal Polyp Tissue in a Biobank for the Isolation and Culture of Primary Epithelial Cells.

Epithelial cells may play an important role in the pathologic process of chronic rhinosinusitis with nasal polyps. Therefore, providing epithelial cells from a biobank could greatly contribute to further research. In the present work, the isolation of epithelial cells from long-term cryopreserved tissue is demonstrated. Polyp tissues were cryopreserved in a commercially available freezing medium with dimethyl sulfoxide and stored in liquid nitrogen. The outgrowth and proliferation of epithelial cells from cryopreserved tissue were evaluated and compared to that of fresh tissue. Flow cytometric analysis with anti-cytokeratin, anti-p63, and anti-Ki-67 was performed to identify epithelial cells and determine differentiation and proliferation. A functionality test was performed by determining type 2-relevant proteins, representatively thymic stromal lymphopoietin (TSLP) and periostin, using ELISA. Primary epithelial cells could be isolated from cryopreserved tissues. Cells from cryopreserved tissues showed comparable outgrowth and proliferation to that of fresh tissue. Isolated epithelial cells showed high cytokeratin, p63, and Ki-67 expression and secreted TSLP and periostin. In the present study, a method for long-term cryopreservation of polyp tissue was established, thereby enabling the isolation and cell culture of primary cell culture at a later time. Epithelial cell availability should be greatly improved by including this method in a biobank.

Comprehensive evaluation of the effects of long-term cryopreservation on peripheral blood mononuclear cells using flow cytometry

Human peripheral blood mononuclear cells (PBMCs) originate from hematopoietic stem cells in the bone marrow, which mainly includes lymphocytes (T cells, B cells, and natural killer cells) and monocytes. Cryopreserved PBMCs providing biobank resources are crucial for clinical application or scientific research. Here, we used flow cytometry to explore the influence of long-term cryopreservation on the quality of PBMCs with the aim of providing important evidence for the effective utilization of biobank resources. The PBMCs were isolated from the peripheral blood, which was collected from volunteers in the hospital. After long-term cryopreservation in liquid nitrogen, we analyzed the changes in cell numbers, viability, and multiple subtypes of PBMCs and studied the apoptosis, proliferation, activation, function, and status of T cells in comparison with freshly isolated PBMCs by flow cytometry, and then further tracked the effects of long-term cryopreservation on the same sample. Although the different cell types in the PBMCs dynamically changed compared with those in the freshly isolated samples, PBMC recovery and viability remained stable after long-term cryopreservation, and the number of most innate immune cells (e.g., monocytes and B cells) was significantly reduced compared to that of the freshly isolated PBMCs or long-term cryopreserved PBMCs; more importantly, the proportion of T cell subtypes, apoptosis, proliferation, and functional T cells, except for Tregs, were not affected by long-term cryopreservation. However, the proportions of activated T, naïve T, central memory T, effector T, and effector memory T cells dynamically changed after long-term cryopreservation. This article provides important evidence for the effective utilization of biobank resources. Long-term cryopreserved PBMCs can be partly used as biological resources for clinical research or basic studies, but the effect of cryopreservation on PBMCs should be considered when selecting cell samples, especially in research relating to activating or inhibiting function.

Experiment-Based Validation of Corneal Lenticule Banking in a Health Authority-Licensed Facility.

With the expected rise in patients undergoing refractive lenticule extraction worldwide, the number of discarded corneal stromal lenticules will increase. Therefore, establishing a lenticule bank to collect, catalog, process, cryopreserve, and distribute the lenticules (for future therapeutic needs) could be advantageous. In this study, we validated the safety of lenticule banking that involved the collection of human lenticules from our eye clinic, transportation of the lenticules to a Singapore Ministry of Health-licensed lenticule bank, processing, and cryopreservation of the lenticules, which, after 3 months or, a longer term, 12 months, were retrieved and transported to our laboratory for implantation in rabbit corneas. The lenticule collection was approved by the SingHealth Centralised Institutional Review Board (CIRB). Both short-term and long-term cryopreserved lenticules, although not as transparent as fresh lenticules due to an altered collagen fibrillar packing, did not show any sign of rejection and cytotoxicity, and did not induce haze or neovascularization for 16 weeks even when antibiotic and steroidal administration were withdrawn after 8 weeks. The lenticular transparency progressively improved and was mostly clear after 4 weeks, the same period when we observed the stabilization of corneal hydration. We showed that the equalization of the collagen fibrillar packing of the lenticules with that of the host corneal stroma contributed to the lenticular haze clearance. Most importantly, no active wound healing and inflammatory reactions were seen after 16 weeks. Our study suggests that long-term lenticule banking is a feasible approach for the storage of stromal lenticules after refractive surgery. Impact statement Since 2011, close to 3 million refractive lenticule extraction procedures have been performed. The majority of the extracted lenticules are discarded. The lenticules could have been cryopreserved and retrieved at a later date for therapeutic or refractive applications. Therefore, establishing a lenticule bank to collect, catalog, process, cryopreserve, and distribute the lenticules could be advantageous. In this study, we simulated a lenticule banking service in a validated health authority-licensed facility and showed that long-term cryopreservation of the lenticules in the facility was safe and feasible in vivo.

High Integrity and Fidelity of Long-Term Cryopreserved Umbilical Cord Blood for Transplantation.

Umbilical cord blood (UCB) is used as a source of donor cells for hematopoietic stem cell (HSC) transplantation. The success of transplantation is dependent on the quality of cord blood (CB) units for maximizing the chance of engraftment. Improved outcomes following transplantation are associated with certain factors of cryopreserved CB units: total volume and total nucleated cell (TNC) count, mononuclear cell (MNC) count, and CD34+ cell count. The role of the storage period of CB units in determining the viability and counts of cells is less clear and is related to the quality of cryopreserved CB units. Herein, we demonstrate the recovery of viable TNCs and CD34+ cells, as well as the MNC viability in 20-year-old cryopreserved CB units in a CB bank (MEDIPOST Co., Ltd., Seongnam-si, Gyeonggi-do, Korea). In addition, cell populations in CB units were evaluated for future clinical applications. The stable recovery rate of the viability of cryopreserved CB that had been stored for up to 20 years suggested the possibility of uses of the long-term cryopreservation of CB units. Similar relationships were observed in the recovery of TNCs and CD34+ cells in units of cryopreserved and fresh CB. The high-viability recovery of long-term cryopreserved CB suggests that successful hematopoietic stem cell (HSC) transplantation and other clinical applications, which are suitable for treating incurable diseases, may be performed regardless of long-term storage.

Long-Term Cryopreservation Does Not Affect Quality of Peripheral Blood Stem Cell Grafts: A Comparative Study of Native, Short-Term and Long-Term Cryopreserved Haematopoietic Stem Cells

Cryopreserved haematopoietic progenitor cells are used to restore autologous haematopoiesis after high dose chemotherapy. Although the cells are routinely stored for a long period, concerns remain about the maximum storage time and the possible negative effect of storage on their potency. We evaluated the effect of cryopreservation on the quality of peripheral stem cell grafts stored for a short (3 months) and a long (10 years) period and we compared it to native products.The viability of CD34+ cells remained unaffected during storage, the apoptotic cells were represented up to 10% and did not differ between groups. The clonogenic activity measured by ATP production has decreased with the length of storage (ATP/cell 1.28 nM in native vs. 0.63 in long term stored products, P < 0.05). Only borderline changes without statistical significance were detected when examining mitochondrial and aldehyde dehydrogenase metabolic activity and intracellular pH, showing their good preservation during cell storage. Our experience demonstrates that cryostorage has no major negative effect on stem cell quality and potency, and therefore autologous stem cells can be stored safely for an extended period of at least 10 years. On the other hand, long term storage for 10 years and longer may lead to mild reduction of clonogenic capacity. When a sufficient dose of stem cells is infused, these changes will not have a clinical impact. However, in products stored beyond 10 years, especially when a low number of CD34+ cells is available, the quality of stem cell graft should be verified before infusion using the appropriate potency assays.

How old is too old? In vivo engraftment of human peripheral blood stem cells cryopreserved for up to 18 years - implications for clinical transplantation and stability programs.

Peripheral blood stem cells (PBSC) are commonly cryopreserved awaiting clinical use for hematopoietic stem cell transplant. Long term cryopreservation is commonly defined as five years or longer, and limited data exists regarding how long PBSC can be cryopreserved and retain the ability to successfully engraft. Clinical programs, stem cell banks, and regulatory and accrediting agencies interested in product stability would benefit from such data. Thus, we assessed recovery and colony forming ability of PBSC following long-term cryopreservation as well as their ability to engraft in NOD/SCID/IL-2Rγnull (NSG) mice. To investigate the in vivo engraftment potential of long-term cryopreserved PBSC units. PBSC units which were collected and frozen using validated clinical protocols were obtained for research use from the Cellular Therapy Laboratory at Indiana University Health. These units were thawed in the Cellular Therapy Laboratory using clinical standards of practice, and the pre-freeze and post-thaw characteristics of the units were compared. Progenitor function was assessed using standard colony-forming assays. CD34-selected cells were transplanted into immunodeficient mice to assess stem cell function. Ten PBSC units with mean of 17 years in cryopreservation (range 13.6-18.3 years) demonstrated a mean total cell recovery of 88% ± 12% (range 68%-110%) and post-thaw viability of 69% ± 17% (range 34%-86%). BFU-E growth was shown in 9 of 10 units and CFU-GM growth in 7 of 10 units post-thaw. Immunodeficient mice were transplanted with CD34-selected cells from four randomly chosen PBSC units. All mice demonstrated long-term engraftment at 12 wk with mean 34% ± 24% human CD45+ cells, and differentiation with presence of human CD19+, CD3+ and CD33+ cells. Harvested bone marrow from all mice demonstrated growth of erythroid and myeloid colonies. We demonstrated engraftment of clinically-collected and thawed PBSC following cryopreservation up to 18 years in NSG mice, signifying likely successful clinical transplantation of PBSC following long-term cryopreservation.

Fidelity of long-term cryopreserved adipose-derived stem cells for differentiation into cells of ocular and other lineages

Adipose-Derived Stem Cells (ADSCs) have an important contribution in regenerative medicine ranging from testing stem cell therapy for disease treatment in pre-clinical models to clinical trials. For immediate use of stem cells for therapy, there is a requirement of the high dose of stem cells at different time points which can be met by cryopreservation. In this study, we evaluated the characteristics of long-term cryopreserved ADSCs and their regenerative potential after an average of twelve-year cryopreservation. Revived ADSCs were examined for cell viability and proliferation by trypan blue, Calcein/Hoechst and MTT assay. Expression of stem cell markers was examined by flow cytometry, immunostaining and qPCR. Colony forming efficiency and spheroid formation ability were also assessed. Multilineage differentiation potential was evaluated by induction into osteocytes, adipocytes, neural cells, corneal keratocytes and trabecular meshwork (TM) cells. Post-thaw, ADSCs maintained expression of stem cell markers CD90, CD73, CD105, CD166, NOTCH1, STRO-1, ABCG2, OCT4, KLF4. ADSCs retained colony and spheroid forming potential. These cells were able to differentiate into osteocytes, confirmed by Alizarin Red S staining and elevated expression of osteocalcin and osteopontin; into adipocytes by Oil Red O staining and elevated expression of PPARγ2. ADSCs could differentiate into neural cells, stained positive to β-III tubulin, neurofilament, GFAP as well as elevated expression of nestin and neurofilament mRNAs. ADSCs could also give rise to corneal keratocytes expressing keratocan, keratan sulfate, ALDH and collagen V, and to TM cells expressing CHI3L1 and AQP1. Differentiated TM cells responded to dexamethasone treatment with increased Myocilin expression, which could be used as in vitro glaucoma model for further studies. Conditioned medium from ADSCs was found to impart a regenerative effect on primary TM cells. In conclusion, ADSCs maintained their stemness and multipotency after long-term cryopreservation with variability between different donors. This study can have great repercussions in regenerative medicine and pave the way for future clinical trials using cryopreserved ADSCs.

What was the fate of human embryos following long-term cryopreservation (≥12 years) and frozen embryo transfer?

Do human embryos survive long-term cryopreservation (CP) (≥12 years) and implant after frozen embryo transfer (ET)? Human embryos remain usable after long-term CP. Several cohort studies have reported the live birth rate or neonatal outcomes of human embryos after CP for up to 5 years. Only a few case reports have described successful live births from human embryos after long-term CP up to 12 years. This retrospective observational study in China included 20 patients (128 embryos) from March 2016 to April 2017. Twenty patients who had at least one live birth during their previous IVF/ICSI treatments and had surplus embryos cryopreserved were observed. Data concerning frozen embryo recovery, pregnancy and obstetric outcomes following frozen ET were recorded. A total of 128 embryos of 20 patients were observed. The embryo storage duration was 12.0-17.1 years, with a mean of 13.9 ± 1.73 years. In all, 115 embryos were thawed to transfer, with a survival rate of 74%. Sixty embryos were further cultured, which resulted in 20 blastocysts with a blastocyst formation rate of 33%. There were 21 cleavage-stage embryos and 13 blastocysts transferred in a total of 12 and 11 cycles, respectively, which resulted in one biochemical pregnancy, one first trimester miscarriage, two ectopic pregnancies, three singletons and one case of twins, with a clinical pregnancy rate of 25% (D3 ET) and 36% (blastocyst transfer) and a live birth rate of 17% (D3 ET) and 27% (blastocyst transfer). Two of the four patients who had live birth developed gestational diabetes mellitus. One of the five live births was a preterm delivery. The sample size was small due to the unique study population, and all the embryos underwent slow freezing. The fate of long-term cryopreserved embryos after vitrification is still unclear. The results provide evidence to support the use of embryos after extended CP to preserve patients' fertility. This study was supported by grants from the National Key Research and Development Programme of China (2016YC1000205) and the Guangzhou Scientific Programme (201508020006). None of the authors has any conflicts of interest to declare.

Ex Vivo-expanded Natural Killer Cells Derived From Long-term Cryopreserved Cord Blood are Cytotoxic Against Primary Breast Cancer Cells.

With over 600,000 units of umbilical cord blood (CB) stored on a global scale, it is important to elucidate the therapeutic abilities of this cryopreserved reservoir. In the advancing field of natural killer (NK) cell cancer immunotherapy, CB has proven to be a promising and noninvasive source of therapeutic NK cells. Although studies have proven the clinical efficacy of using long-term cryopreserved CB in the context of hematopoietic stem cell transplantations, little is known about its use for the ex vivo expansion of effector immune cells. Therefore, our group sought to derive ex vivo-expanded NK cells from long-term cryopreserved CB, using an artificial antigen presenting cell-mediated expansion technique. We compared the expansion potential and antitumor effector function of CB-derived NK (CB-NK) cells expanded from fresh (n=4), short-term cryopreserved (<1-year old, n=5), and long-term cryopreserved (1-10-year old, n=5) CB. Here, we demonstrated it is possible to obtain an exponential amount of expanded CB-NK cells from long-term cryopreserved CB. Ex vivo-expanded CB-NK cells had an increased surface expression of activating markers and showed potent antitumor function by producing robust levels of proinflammatory cytokines, interferon-γ, and tumor necrosis factor-α. Moreover, expanded CB-NK cells (n=3-5) demonstrated cytotoxicity towards primary breast cancer cells (n=2) derived from a triple-negative breast cancer and an estrogen receptor-positive/progesterone receptor-positive breast cancer patient. Long-term cryopreservation had no effect on the expansion potential or effector function of expanded CB-NK cells. Therefore, we propose that long-term cryopreserved CB remains clinically useful for the ex vivo expansion of therapeutic NK cells.

Cryopreservation (-20°C) of canine corneoscleral tissue: histological, microbiological, and ultrastructural study.

To evaluate microbiological, histological, and ultrastructural characteristics of short-term cryopreserved (STC) canine corneoscleral tissue (<1year) and to compare it with long-term cryopreserved (LTC) tissue (>6years). Thirty-six healthy canine globes. After a decontamination protocol, globes were enucleated and stored at -20°C. Corneoscleral tissue was evaluated at different periods: <1year (20 eyes) and >6years (12 eyes). Four eyes were used as controls. Microbiologic study included direct (blood, McConkey and Sabouraud agars) and enrichment (brain-heart infusion broth) cultures. Cryopreservation artifacts were evaluated by hematoxylin-eosin. Corneoscleral collagen organization and number of normal and dead keratocytes were established by transmission electron microscopy (TEM). Terminal deoxynucleotidyl transferasedUTP nick end labeling (TUNEL) was also used for keratocyte characterization. Corneal microbial growth was observed in 25% of the direct STC cultures, and in 47.4% and 16.7% of the enriched STC and LTC cultures, respectively. Scleral STC direct cultures were 30% positive, while enrichment cultures were positive in 66.7% and 16.7% of the STC and LTC, respectively (P=0.011). Cryopreservation artifacts were higher in LTC tissues (P<0.001). Apoptotic keratocytes were predominant by TEM and TUNEL, in both STC and LTC. Minimal structural differences were detected in collagen organization between STC and LTC. Cryopreservation of canine corneoscleral tissue seems to reduce bacterial contamination over time. Apoptosis is the main way of death of cryopreserved canine keratocytes. Based on the lack of significant structural differences between STC and LTC samples, these cryopreserved tissues could potentially be used for tectonic support for at least 8years without structural or microbiological impediment.

Long-term culture-induced phenotypic difference and efficient cryopreservation of small intestinal organoids by treatment timing of Rho kinase inhibitor.

AIMTo investigate a suitable long-term culture system and optimal cryopreservation of intestinal organoid to improve organoid-based therapy by acquiring large numbers of cells.METHODSCrypts were isolated from jejunum of C57BL/6 mouse. Two hundred crypts were cultured in organoid medium with either epidermal growth factor/Noggin/R-spondin1 (ENR) or ENR/CHIR99021/VPA (ENR-CV). For subculture, organoids cultured on day 7 were passaged using enzyme-free cell dissociation buffer (STEMCELL Technologies). The passage was performed once per week until indicated passage. For cryopreservation, undissociated and dissociated organoids were resuspended in freezing medium with or without Rho kinase inhibitor subjected to different treatment times. The characteristics of intestinal organoids upon extended passage and freeze-thaw were analyzed using EdU staining, methyl thiazolyl tetrazolium assay, qPCR and time-lapse live cell imaging.RESULTSWe established a three-dimensional culture system for murine small intestinal organoids using ENR and ENR-CV media. Both conditions yielded organoids with a crypt-villus architecture exhibiting Lgr5+ cells and differentiated intestinal epithelial cells as shown by morphological and biochemical analysis. However, during extended passage (more than 3 mo), a comparative analysis revealed that continuous passaging under ENR-CV conditions, but not ENR conditions induced phenotypic changes as observed by morphological transition, reduced numbers of Lgr5+ cells and inconsistent expression of markers for differentiated intestinal epithelial cell types. We also found that recovery of long-term cryopreserved organoids was significantly affected by the organoid state, i.e., whether dissociation was applied, and the timing of treatment with the Rho-kinase inhibitor Y-27632. Furthermore, the retention of typical morphological characteristics of intestinal organoids such as the crypt-villus structure from freeze-thawed cells was observed by live cell imaging.CONCLUSIONThe maintenance of the characteristics of intestinal organoids upon extended passage is mediated by ENR condition, but not ENR-CV condition. Identified long-term cryopreservation may contribute to the establishment of standardized cryopreservation protocols for intestinal organoids for use in clinical applications.

Cryopreservation (−20 °C) of feline corneoscleral tissue: histologic, microbiologic, and ultrastructural study

To evaluate microbiological, histologic, and ultrastructural characteristics of short-term cryopreserved (STC) feline corneoscleral tissue (<1 year) and to compare it with long-term cryopreserved (LTC) tissue (>7 years). Twenty healthy feline globes were obtained from 2003 to 2013. After a decontamination protocol, globes were enucleated and stored at -20 °C in broad-spectrum antibiotics. Corneoscleral tissue was evaluated at different storage periods: <1 year (10 eyes) and >7 years (8 eyes). Two eyes were used as controls. Microbiologic study included direct (blood, McConkey, and Sabouraud agars) and enrichment (brain-heart infusion broth) cultures. Cryopreservation artifacts were evaluated by hematoxylin-eosin. Corneoscleral collagen organization and number of normal and dead keratocytes were established by transmission electron microscopy. Although microbiologic cultures were positive only in STC [direct (20.8%); enrichment (37.5%)], significant differences between periods were only found in enrichment cultures (P = 0.006). Cryopreservation artifacts were most commonly observed in LTC tissues (P < 0.001). Normal keratocytes were predominant in STC corneas (STC 58.3%, LTC 12.5%) and apoptotic ones in LTC (STC 41.7%, LTC 75%), whereas necrotic keratocytes were only seen in LTC (LTC 12.5%) (P = 0.046). No structural differences were detected in collagen organization between STC and LTC (Pcornea = 0.147; Psclera = 0.362). Cryopreservation of feline corneoscleral tissue seems to reduce bacterial contamination over time. Apoptosis is the main cause of death of cryopreserved feline keratocytes. Based on the lack of significant structural differences between STC and LTC samples, these cryopreserved tissues could potentially be used for tectonic support for at least 10 years without structural or microbiological impediment.

Phenotypic and functional characterization of long-term cryopreserved human adipose-derived stem cells.

Cryopreservation represents an effective technique to maintain the functional properties of human adipose-derived stem cells (ASCs) and allows pooling of cells via long-term storage for clinical applications, e.g., cell-based therapies. It is crucial to reduce freezing injury during the cryopreservation process by loading the ASCs with the optimum concentration of suitable cryoprotective agents (CPAs). In this study, human ASCs were preserved for 3 months in different combinations of CPAs, including 1) 0.25 M trehalose; 2) 5% dimethylsulfoxide (DMSO); 3) 10% DMSO; 4) 5% DMSO + 20% fetal bovine serum (FBS); 5) 10% DMSO + 20% FBS; 6) 10% DMSO + 90% FBS. Interestingly, even with a reduction of DMSO to 5% and without FBS, cryopreserved ASCs maintained high cell viability comparable with standard cryomedium (10% DMSO + 90% FBS), with normal cell phenotype and proliferation rate. Cryopreserved ASCs also maintained their differentiation capability (e.g., to adipocytes, osteocytes and chondrocytes) and showed an enhanced expression level of stemness markers (e.g., NANOG, OCT-4, SOX-2 and REX-1). Our findings suggest that 5% DMSO without FBS may be an ideal CPA for an efficient long-term cryopreservation of human ASCs. These results aid in establishing standardized xeno-free long-term cryopreservation of human ASCs for clinical applications.

Evaluation of Canine Bone Marrow-derived Mesenchymal Stem Cells After Long-term Cryopreservation

Autologous bone marrow-derived mesenchymal stem cell (BMSCs)-based therapies show great potential in regenerative medicine. However, long-term storage and preservation of BMSCs for clinical use is still a great clinical challenge. The present study aimed to analyze the effect of long-term cryopreservation on the regenerative ability of BMSCs. After cryopreservation of BMSCs from beagle dogs for three years, cell viability, and quantitative analysis of alkaline phosphatase (ALP) activity, surface adherence, and mineralized nodule formation were analyzed. BMSCs in cell-scaffold complex were then implanted into nude mice. There was no significant difference in cell viability and ALP activity between osteogenic differentiation and non-osteogenic differentiation of BMSCs, and BMSCs in cell-scaffold complex retained osteogenic differentiation ability in vivo. These results indicate that long-term cryopreserved BMSCs maintain their have capacity to contribute to regeneration.

Assessment of long-term cryopreservation for production of hCTLA4Ig in transgenic rice cell suspension cultures

For the commercialization of plant-made pharmaceuticals (PMPs) using transgenic plant cell cultures, the establishment of a cell-banking system has been known to be an essential process. Plant cells are traditionally maintained by repeated subcultures. However, this method has several problems including genetic instability of transformed cell lines, time- and cost-consuming. In this study, long-term cryopreserved rice suspension cells were firstly investigated for the production of human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig). The cryopreserved cells for 5 years were regrowed to callus successfully and then suspended into the liquid medium. Consequently, the maximum cell mass and the hCTLA4Ig production were similar levels compared to those of the non-cryopreserved cells (control) even though hCTLA4Ig productivity was 1.7-fold higher than that of control. To further assess the level of improvements in hCTLA4Ig productivity in cryopreserved cells, hCTLA4Ig production profiles were statistically assessed between data of the cryopreserved cells for 5 years and annual data of non-cryopreserved cells maintained by subculture for 5 years. These results also indicate that hCTLA4Ig productivity in cryopreserved cells for 5 years was significantly increased (p-value: <0.001, 95% confidence interval) and it could be related to cell lysis resulting in release of hCTLA4Ig which was confirmed by the measurement of electrolyte leakage. In conclusion, we show that the long-term cryopreservation of transgenic rice cells was possible to support stable cell lines for the production of PMPs.

Long term cryopreservation in 5% DMSO maintains unchanged CD34+ cells viability and allows satisfactory hematological engraftment after peripheral blood stem cell transplantation

Peripheral blood stem cell cryopreservation is associated with cell damage and decreased viability. We evaluated the impact of up to 10years of cryopreservation (5% DMSO) on viability of CD34(+) cells utilizing graft samples of consecutive patients (2002-2012) with different malignancies who underwent stem cell collection and transplantation. Viability of CD34(+) cells from oncohaematological patients measured after 5weeks (97·2±0·6%) or after 9-10years of cryopreservation (95·9±0·5%) was unaffected. Haemoglobin, granulocyte and platelet recovery after transplantation of long-term cryopreserved grafts occurred within 8-13days. CD34(+) stem cells can be safely stored up to 9-10years, without affecting cell viability and clinical effectiveness.

Quality of long-term cryopreserved umbilical cord blood units for hematopoietic cell transplantation

The aim of this study was to evaluate the quality of long-term cryopreserved umbilical cord blood (CB) units for hematopoietic cell transplantation (HCT). The recovery of the number of total nucleated cell (TNC), hematopoietic progenitor cells (HPCs; CD34+ cells, colony-forming units-granulocyte/macrophages [CFU-GMs]), and the percentage of viable cells, CD34+ CD38- cells, and CD34+ CXCR4+ cells of CB units cryopreserved for 10years for HCT were examined. Eighteen CB units cryopreserved for 10years (as the study group) and for 1month (as the control group), respectively, were analyzed. The recovery rate of TNC, CD34+ cells and CFU-GMs were 88.72±16.40, 68.39±18.37 and 42.28±38.16% for the study group and 80.17±14.46, 72.67±20.38 and 49.61±36.39% for the control group (p=0.106, p=0.513 and p=0.559, respectively). There were no significant differences in the recovery rate of TNC, CD34+ cells and CFU-GMs between the study group and the control group. The mean basal percentage of viable cells, CD34+ CD38- cells, and CD34+ CXCR4+ cells after thawing were 83.69±9.45, 9.11±4.13 and 81.65±10.82% for the study group. These results indicate that long-term cryopreservation does not negatively affect the quality of CB units for HCT.