Robert G. Van Buskirk

Cell viability improves following inhibition of cryopreservation-induced apoptosis.

SummaryA new concept in cryopreservation solution design was developed that focuses on the use of an intracellular-type, hypothermic maintenance medium coupled with additives that inhibit cryopreservation-induced apoptosis. Hypo-Thermosol® (HTS), a hypothermic (4° C) maintenance medium utilized in the long-term storage of cell, tissue, and organ systems, was tested for cryoprotective capability on a renal cell line (Madin-Darby Canine Kidney cells). HTS and HTS derivatives were tested against conventional cell culture medium (Dulbeccos Minimal Essential medium, DME) as the cryoprotectant carrier solution because (1) cells are exposed to an extended state of hypothermia during the freeze-thaw process, and (2) HTS is designed to protect cells exposed to a hypothermic state. Cells separately cryopreserved in either HTS or DME +5% dimethyl sulfoxide (DMSO) yielded equivalent 24-h postthaw survival (∼30%) and 5-d recovery (∼90%). Cells cryopreserved in CryoStor® CS 5, a HTS derivative containing 5% DMSO, yielded ∼75% 24-h postthaw survival and recovery to 100% within 3 d. DNA gel electrophoresis was performed to determine the mechanisms of cell death contributing to cryopreservation failure. Cells preserved in DME (DMSO-free) died primarily through necrosis, whereas cells preserved in either DME +5% DMSO, HTS, or CryoStor® CS 5 died through a combination of apoptosis and necrosis. This observation led to the inclusion of an apoptotic inhibitor designed to improve cryopreservation outcome. MDCK cells cryopreserved in CryoStor® CS 5 supplemented with an apoptotic inhibitor (Caspase I Inhibitor V), hereafter termed CryoStor® CS 5N, resulted in a 24-h postthaw survival and recovery rate exceeding that of any other cryoprotective solution tested (85%). We conclude that: (1) the use HTS (a dextran-based, intracellular-type solution), without DMSO can yield postthaw viability equivalent to that of standard DMSO-based cryopreservation methods, (2) postthaw viability can be significantly increased through the use of an intracellular-type solution in conjunction with DMSO, (3) the use of HTS allows for cryopreservation to be accomplished with reduced levels of cryoprotectants, and (4) the regulation of apoptosis is essential for the improvement of cryopreservation outcome.

Gene Activation of the Apoptotic Caspase Cascade Following Cryogenic Storage

The improvement in preservation is now critical for support of the newly emerging fields of tissue engineering and regenerative medicine. The expanding number of complex biologics banked for therapeutic applications necessitates the development of new preservation technologies. Recent studies in biologic preservation have implicated apoptosis as a contributing factor to cell death. These studies have described the role and extent of apoptotic cell death following cryopreservation but have not addressed investigation into the signaling pathways responsible for program execution. Accordingly, we investigated the role of apoptotic gene activation of caspase3 at the genetic and protein levels following cryopreservation (culture media + dimethyl sulfoxide) in a human fibroblast cell model. Additionally, investigation into the extent of caspase-3 involvement in cell death following preservation in a newly developed cryopreservation medium was performed. In this study we report on the upregulation of transcripti...

Microporosity of the substratum regulates differentiation of MDCK cells in vitro

SummaryWe have analyzed the ability of the physical substratum to modulate both the ultrastructural and protein synthetic characteristics of the Madin-Darby canine kidney (MDCK) renal cell line. When MDCK cells were seeded on Millipore Millicell CM microporous membrane cell culture inserts they demonstrated a more columnar organization with an increase in cell density sixfold greater than the same cells seeded on conventional plastic substrata. After 1 wk postseeding on the microporous membrane a partial basal lamina was noted, with a contiguous basement membrane being apparent after 2 wk. One-dimensional sodium dodecyl sulfate gel electrophoresis was used to analyze detergent-solubilized proteins from MDCK cells maintained on plastic substrata vs. microporous membranes. When proteins were pulse-labeled with [35S]methionine, a 55 kDa protein was evident in the cytosolic extract of cells grown on collagen, laminin, and nontreated plastic substrata; but this labeled protein was not evident in similar extracts from cells grown on collagen and laminin-coated microporous membranes. To test if the polarized, basement-membrane secreting phenotype of the MDCK cells could be generated on a microporous membrane without pretreatment with any extracellular matrix (ECM) components, cells were seeded on the Millipore Millicell HA (cellulosic) microporous membrane. This type of substrata does not need a coating of ECM components for cell attachment. A partial basement membrane was formed below cells where the basal surface of the cell was planar, but not in areas where the cell formed large cytoplasmic extensions into the filter. This led us to the conclusion that the microporous nature of the substrata can dictate both ultrastructural and protein synthetic activities of MDCK cells. Furthermore, we suggest that both the planar nature of the basal surface and the microporosity of the substrate are corequisites for the deposition of the basement membrane.

Improved Hypothermic Preservation of Human Renal Cells Through Suppression of Both Apoptosis and Necrosis

A new platform of hypothermic solutions, the HypoThermosol® (HTS) series, has been developed for the improved hypothermic storage of cells, tissues, and organs. Cells and tissues cold-stored in HTS-FRS demonstrate improved viability when returned to normothermic temperatures in comparison with the parent solution, HTS-BASE, or University of Wisconsin (UW) solution (UW-ViaSpan®). While our group and others have implicated apoptosis as a major player in cell death initiated by extended hypothermic storage, it has been unclear if the improved performance of HTS-FRS as a hypothermic storage solution is due to its ability to inhibit apoptosis. Data reported herein show that human renal cells hypothermically stored in renal cell culture medium, HTS-FRS, HTS-BASE, or UW solution demonstrated improved survival in HTS-FRS. Following 5 days of hypothermic preservation and 1 day of recovery at 37°C, cells preserved in HTS-FRS exhibit 75% metabolic activity, whereas cells stored in HTS-BASE, UW, or culture media demo...

A Human Epidermal Model can be Assayed Employing a Multiple Fluorescent Endpoint Assay and the Cytofluor 2300

AbstractA human epidermal model (HEM) was developed that could be rapidly and automatically assayed in the CytoFluor 2300 (Millipore Corporation, Bedford, MA) spectrofluorometer using multiple site-and activity-specific fluorescent probes. The HEM was cultured on the optically translucent Millipore Millicell CM microporous membrane. Application of a variety of fluorescent dyes to this membrane without the HEM revealed negligible nonspecific dye association. The HEM was differentiated on a cross-linked collagen matrix and the latter was also found to retain less dye than the HEM. Feasibility experiments using the site/activity-specific dyes calcein-AM (plasma membrane integrity indicator), sodium fluorescein (epidermal permeability indicator), 5-chloromethylfluorescein diacetateacetoxymethyl ester (CMFDA-AM; intracellular glutathione level indicator), rhodamine 123 (mitochondrial activity indicator), neutral red (lysosomal integrity indicator), Fluo3-AM (intracellular calcium indicator), and a similar huma...

The unfolded protein response in human corneal endothelial cells following hypothermic storage: Implications of a novel stress pathway ☆

Human corneal endothelial cells (HCEC) have become increasingly important for a range of eye disease treatment therapies. Accordingly, a more detailed understanding of the processing and preservation associated stresses experienced by corneal cells might contribute to improved therapeutic outcomes. To this end, the unfolded protein response (UPR) pathway was investigated as a potential mediator of corneal cell death in response to hypothermic storage. Once preservation-induced failure had begun in HCECs stored at 4°C, it was noted that necrosis accounted for the majority of cell death but with significant apoptotic involvement, peaking at several hours post-storage (4-8h). Western blot analysis demonstrated changes associated with apoptotic activation (caspase 9, caspase 3, and PARP cleavage). Further, the activation of the UPR pathway was observed through increased and sustained levels of ER folding and chaperone proteins (Bip, PDI, and ERO1-Lα) in samples experiencing significant cell death. Modulation of the UPR pathway using the specific inhibitor, salubrinal, resulted in a 2-fold increase in cell survival in samples experiencing profound cold-induced failure. Furthermore, this increased cell survival was associated with increased membrane integrity, cell attachment, and decreased necrotic cell death populations. Conversely, addition of the UPR inducer, tunicamycin, during cold exposure resulted in a significant decrease in HCEC survival during the recovery period. These data implicate for the first time that this novel cell stress pathway may be activated in HCEC as a result of the complex stresses associated with hypothermic exposure. The data suggest that the targeted control of the UPR pathway during both processing and preservation protocols may improve cell survival and function of HCEC thus improving the clinical utility of these cells as well as whole human corneas.

Development of a tissue engineered human prostate tumor equivalent for use in the evaluation of cryoablative techniques

The study of the effectiveness of cryotherapy as a curative treatment for prostate cancer has often relied on the use of either in vitro cell culture monolayers or animal models. While the data gleaned from these studies have been valuable, each model has inherent limitations. In order to bridge the gap between in vitro studies and clinical applications, we developed a 3-dimensional, tissue engineered human prostate cancer model to simulate and assess the effects of cryotherapy and adjunctive treatments on cell viability and activation of cell death pathways throughout the thermally variable freeze zone. Human prostate cancer cells (PC3) were seeded into collagen based matrices and cryolesions were generated using an Oncura SeedNet® Gold cryosurgical device with 17-gauge cryoprobes. Analyses revealed widespread necrosis diminishing towards the edge of the freeze zone, and a time-dependent wave of apoptosis starting as early as 1 hr post-thaw at low temperatures (< −40°C) and moving toward the periphery (−20°C) as recovery times reached 12 and 24 hr. Distal to the −10°C isotherm, minimal cell death was apparent (< 20%) over controls. The adjunctive use of chemotherapeutic agents in conjunction with cryosurgery displayed a similar induction of cell death cascades, but with the zone of cryodestruction extending ~10 to 15°C further into the freeze zone periphery. By providing an extracellular environment and a matrix to minimize innate variables, the tissue engineered model yielded a more in vivo-like, tumor-like environment supportive of a deeper understanding of the specific biological responses of cancer cells/tumors to cryotherapeutic intervention.

Cold-Storage of Synthetic Human Epidermis in HypoThermosol

There is a growing need for engineered tissues in a wide variety of medical applications and as alternatives to animal tissues for in vitro toxicology testing. While techniques for the preparation of a variety of synthetic tissue constructs have been devised, little attention has been focused upon the optimum conditions necessary for storage and shipping of these tissue devices. This study investigates the effects of hypothermic storage on synthetic human epidermis (EpiDerm, MatTek Corp., Ashland, MA) and specifically examines the quality of storage in keratinocyte growth medium (KGM), a standard skin culture medium, compared with storage in HypoThermosol, a new hypothermic preservation solution. EpiDerm samples were immersed in HypoThermosol for 1 to 13 days at 4 degrees C and were assayed using the noninvasive, viability indicator dye, Alamar Blue (AB). Samples immersed for 1 to 9 days in HypoThermosol retained their viability subsequent to warming to 37 degrees C and for at least 7 days thereafter in culture. During this time samples previously stored in HypoThermosol continued to generate a stratum corneum and their ultrastructural characteristics were similar to EpiDerm that were not exposed to hypothermic solutions. This profile, however, was not apparent in EpiDerm maintained for 1 to 13 days in KGM and subsequently warmed. These samples appeared viable immediately upon warming in most cases, but viability was not retained thereafter. EpiDerm maintained in KGM and allowed to recover at 37 degrees C appeared necrotic and failed to continue to differentiate. The conclusions of this study are the following: (1) HypoThermosol protects the viability of EpiDerm during cold-storage, (2) HypoThermosol preserves EpiDerms ability to differentiate subsequent to warming, and (3) the inferior preservation of samples stored in KGM was most apparent 24 h after warming.

Characterization and modulation of human mesenchymal stem cell stress pathway response following hypothermic storage.

Human mesenchymal stem cell (hMSC) research has grown exponentially in the last decade. The ability to process and preserve these cells is vital to their use in stem cell therapy. As such, understanding the complex, molecular-based stress responses associated with biopreservation is necessary to improve outcomes and maintain the unique stem cell properties specific to hMSC. In this study hMSC were exposed to cold storage (4°C) for varying intervals in three different media. The addition of resveratrol or salubrinal was studied to determine if either could improve cell tolerance to cold. A rapid elevation in apoptosis at 1h post-storage as well as increased levels of necrosis through the 24h of recovery was noted in samples. The addition of resveratrol resulted in significant improvements to hMSC survival while the addition of salubrinal revealed a differential response based on the media utilized. Decreases in both apoptosis and necrosis together with decreased cell stress/death signaling protein levels were observed following modulation. Further, ER stress and subsequent unfolded protein response (UPR) stress pathway activation was implicated in response to hMSC hypothermic storage. This study is an important first step in understanding hMSC stress responses to cold exposure and demonstrates the impact of targeted molecular modulation of specific stress pathways on cold tolerance thereby yielding improved outcomes. Continued research is necessary to further elucidate the molecular mechanisms involved in hypothermic-induced hMSC cell death. This study has demonstrated the potential for improving hMSC processing and storage through targeting select cell stress pathways.

Cellular Components of the Coronary Vasculature Exhibit Differential Sensitivity to Low Temperature Insult

In the treatment of atherosclerosis, therapeutic angioplasty is commonly used to restore normal blood flow in stenosed arteries. This procedure, however, is associated with vascular endothelial cell erosion (denudation) of the intimal lining and subsequent acute arterial closure through restenosis, which involves the encroachment of vascular smooth muscle cells from the medial layer into the lumen. In the vasculature of the heart, coronary artery endothelial cells (CAECs) and coronary artery smooth muscle cells (CASMCs) are major players in the pathogenesis of atherosclerosis in the vasculature of the heart. Interactions between these cell types and the innate immune response are paramount to atherosclerosis and post-intervention complications. The objective of this study was to investigate/optimize the efficacy of cryotherapy (the endovascular application of low temperature) as an adjunctive therapeutic intervention in association with angioplasty to combat restenosis. We report on an in vitro model for ...