Osteoblast viability and differentiation with Me2SO as cryoprotectant compared to osteoblasts from fresh human iliac cancellous bone

The aim of this study was to compare the viability of human osteoblasts cryopreserved with Me2SO to that of fresh human iliac cancellous bone using cell culture techniques. Osteoblasts were obtained by spontaneous outgrowth of human iliac cancellous bone specimens in experiment I. In experiment II, human iliac cancellous bone was frozen with 10% Me2SO at -80 degrees C for 2 weeks and osteoblasts grew spontaneously after thawing at 37 degrees C by removing Me2SO with sucrose. The cells were grown in culture flasks containing DMEM as a culture medium, supplemented with 10% fetal calf serum. They were kept at 37 degrees C in a humidified atmosphere of 95% air and 5% CO2. Cells from the second passage were plated at a density of 5 times 10(3) cells/cm2 in 24-well plates. For detection of viability and differentiation, WST-1 assay, determination of alkaline phosphatase activity, concentration of procollagen I peptide, concentration of osteocalcin, and indirect immunofluorescence for osteopontin, collagen type I, integrin beta1, and fibronectin were applied. Experiments were conducted at four stages of confluence (days 4, 7, 14, and 21 after plating the cells). Based on the results of this study, we conclude that osteoblast-like cells survived cryopreservation and synthesized a range of markers that were consistent with this cell type.     

Preservation of rat bone marrow with dimethylsulphoxide at -150 degrees C.

The authors tested preserving properties of three concentrations of dimethylsulphoxide (15%, 10% and 7.5%) in preservation of rat bone marrow cells at -150 degrees C. Cells of rat bone marrow were frozen at 1 degree C/min to -20 degrees C, 5 degrees C/min to -80 degrees C and then placed directly at -150 degrees C and held at such temperature for 6 months. Vitality of cells was checked monthly for a period of 6 months by means of several vitality tests with dyes (eosin and trypane blue), autoradiography and erythrophagocytosis. It was found that cells capable of cleavage could be equally preserved at such low temperature with all the three DMSO concentrations while mature cells (granulocytes, reticular cells) revealed considerably higher erythrophagocytic activity when preserved at 15% DMSO and lower activity at 10% and 7.5% DMSO.     

Temperature-dependence of the proliferation of mouse bone marrow cells cultured in H2O- and D2O-media.

In an attempto to optimize and standardize the in vitro culture conditions of mouse bone marrow cells for assaying growth regulating factors, we studied the effects of incubation at low temperatures and of a nutrient medium containing deuteriumoxide instead of water. It was found that (1) the proliferative capacity of the cells is significantly increased by pre-incubation for 1-2 h at 0 degrees C rather than at 37 degrees C, measured by both a colony-forming and a 3H-thymidine (3H-tdr) uptake assay. A similar temperature effect on the colony-forming and 3H-tdr uptake ability is apparent after pre-incubation in D2O-medium, yet significantly lower than in H2O-medium. It was concluded that the previously observed protective effects of D2O on ascites tumor cell proliferation and viability and for hemolysis of human erythrocytes is not apparent in proliferating and colony-forming mouse bone marrow cells in vitro.     

Cryopreservation by slow cooling with DMSO diminished production of Oct-4 pluripotency marker in human embryonic stem cells

We tested a "standard" cryopreservation protocol (slow cooling with 10% DMSO) on the human embryonic stem cell (hESC) line H9 containing an Oct-4 (POU5F1) promoter-driven, enhanced green fluorescent protein (EGFP) reporter to monitor maintenance of pluripotency. Cells were cooled to -80 degrees C in cryovials and then transferred to a -80 degrees C freezer. Cells were held at -80 degrees C for 3 days ("short-term storage") or 3 months ("long-term storage"). Vials were thawed in a +36 degrees C water bath and cells were cultured for 3, 7, or 14 days. Propidium iodide (PI) was used to assess cell viability by flow cytometry. Control cells were passaged on the same day that the frozen cells were thawed. The majority of cells in control hESC cultures were Oct-4 positive and almost 99% of EGFP+ cells were alive as determined by exclusion of PI. In contrast, the frozen cells, even after 3 days of culture, contained only 50% live cells, and only 10% were EGFP-positive. After 7 days in culture, the proportion of dead cells decreased and there was an increase in the Oct-4-positive population but microscopic examination revealed large patches of EGFP-negative cells within clusters of colonies even after 14 days of culturing. After 3 months of storage at -80 degrees C the deleterious effect of freezing was even more pronounced: the samples regained a quantifiable number of EGFP-positive cells only after 7 days of culturing following thawing. It is concluded that new protocols and media are required for freezing hESC and safe storage at -80 degrees C as well as studies of the mechanisms of stress-related events associated with cell cryopreservation.     

Effect of culture temperature on erythropoietin production and glycosylation in a perfusion culture of recombinant CHO cells

To investigate the effect of culture temperature on erythropoietin (EPO) production and glycosylation in recombinant Chinese hamster ovary (CHO) cells, we cultivated CHO cells using a perfusion bioreactor. Cells were cultivated at 37 degrees C until viable cell concentration reached 1 x 10(7) cells/mL, and then culture temperature was shifted to 25 degrees C, 28 degrees C, 30 degrees C, 32 degrees C, 37 degrees C (control), respectively. Lowering culture temperature suppressed cell growth but was beneficial to maintain high cell viability for a longer period. In a control culture at 37 degrees C, cell viability gradually decreased and fell below 80% on day 18 while it remained over 90% throughout the culture at low culture temperature. The cumulative EPO production and specific EPO productivity, q(EPO), increased at low culture temperature and were the highest at 32 degrees C and 30 degrees C, respectively. Interestingly, the cumulative EPO production at culture temperature below 32 degrees C was not as high as the cumulative EPO production at 32 degrees C although the q(EPO) at culture temperature below 32 degrees C was comparable or even higher than the q(EPO) at 32 degrees C. This implies that the beneficial effect of lowering culture temperature below 32 degrees C on q(EPO) is outweighed by its detrimental effect on the integral of viable cells. The glycosylation of EPO was evaluated by isoelectric focusing, normal phase HPLC and anion exchange chromatography analyses. The quality of EPO at 32 degrees C in regard to acidic isoforms, antennary structures and sialylated N-linked glycans was comparable to that at 37 degrees C. However, at culture temperatures below 32 degrees C, the proportions of acidic isoforms, tetra-antennary structures and tetra-sialylated N-linked glycans were further reduced, suggesting that lowering culture temperature below 32 degrees C negatively affect the quality of EPO. Thus, taken together, cell culture at 32 degrees C turned out to be the most satisfactory since it showed the highest cumulative EPO production, and moreover, EPO quality at 32 degrees C was not deteriorated as obtained at 37 degrees C.     

TAK-778 enhances osteoblast differentiation of human bone marrow cells via an estrogen-receptor-dependent pathway

TAK-778, a derivative of ipriflavone, has been shown to induce bone growth in in vitro and in vivo models. However, there are no studies evaluating by which mechanism TAK-778 exerts its effect. Considering the evidences that its precursors act via classical estrogen-receptor (ER)-mediated signaling, in the present study, we tested the hypothesis that TAK-778 induces osteogenesis in human bone marrow cell culture via an ER-dependent pathway. Cells were cultured in 24-well culture plates at a cell density of 2 x 10(4) cells/well in culture medium containing: TAK-778 (10(-5) M), Tamoxifen (10(-5) M), TAK-778 (10(-5) M) + Tamoxifen (10(-5) M), and vehicle. During the culture period, cells were incubated at 37 degrees C in a humidified atmosphere of 5% CO(2) and 95% air. At 7, 14, and 21 days, cell proliferation, cell viability, total protein content, alkaline phosphatase (ALP) activity, and bone-like formation were evaluated. Data were compared by two-way ANOVA and Duncan's multiple range test. TAK-778 did not affect cell viability. Cell number was reduced by TAK-778. Total protein content, ALP activity, and bone-like formation were increased by TAK-778. In general, Tamoxifen did not have any effect on cell behavior. However, when cells were cultured in medium containing both TAK-778 and Tamoxifen, the effect of TAK-778 on osteoblast differentiation was inhibited. The present results show that TAK-778 enhances osteoblast differentiation in human bone marrow cell culture, at least in part, via an ER-dependent pathway, since its effect was inhibited by Tamoxifen, a well-known estrogen receptor antagonist.     

TAK-778 enhances osteoblast differentiation of human bone marrow cells

TAK-778 has been shown to induce bone growth in in vitro and in vivo models. However, there are no studies evaluating the effect of TAK-778 on human cells. Thus, the aim of this study was to investigate osteogenesis induced by TAK-778 on human bone marrow cells. Cells were cultured in 24-well culture plates at a cell density of 2 x 10(4) cells/well in culture medium containing TAK-778 (10(-7), 10(-6), and 10(-5) M, each) or vehicle. During the culture period, cells were incubated at 37 degrees C in a humidified atmosphere of 5% CO(2) and 95% air. For attachment evaluation, cells were cultured for 4 and 24 h. After 7, 14, and 21 days, cell proliferation, cell viability, total protein content, alkaline phosphatase (ALP) activity, and bone-like formation were evaluated. Data were compared by ANOVA and Duncan's multiple range test. TAK-778 did not affect cell attachment and viability. Cell number was reduced by TAK-778 in all time period evaluated in a dose-dependent way. The effect of TAK-778 on total protein content, ALP activity and bone-like formation was a dose-dependent increase. The present results suggest that initial cell events such as cell attachment are not affected by TAK-778 while events that indicate osteoblast differentiation including reduced cell proliferation, and increased both ALP activity and bone-like formation are enhanced by TAK-778 in a time and dose-dependent way. It means that TAK-778 could be a useful drug to enhance new bone formation in clinical situations that require rapid restoration of physiologic function, such as orthopedic and maxillofacial surgery.     

Overexpression of the MnSOD transgene product protects cryopreserved bone marrow hematopoietic progenitor cells from ionizing radiation

We determined whether manganese superoxide dismutase (MnSOD)-plasmid liposome (PL) transfection of C57BL/ 6NHsd mouse bone marrow protected cells irradiated at room temperature (24 degrees C) or in the cryopreserved state. MnSOD-overexpressing hematopoietic progenitor 2C6 cells were radioresistant compared to the parent 32D cl 3 cells when irradiated frozen or at 24 degrees C. Fresh whole marrow from mice injected intravenously with MnSOD-PL prior to explant as well as explanted marrow single cell suspensions transfected in vitro were irradiated at 24 degrees C or -80 degrees C. In vivo or in vitro transfection of marrow with MnSOD-PL produced significant radiation protection of irradiated marrow progenitor cells compared to controls at 24 degrees C or -80 degrees C. (in vivo transfection D(0) 2.19 +/- 0.21 at 24 degrees C, D(0) 2.10 +/- 0.07 at -80 degrees C compared to control D(0) 1.56 +/- 0.06 or 1.66 +/- 0.04, P = 0.047 and 0.017 respectively; in vitro transfection D(0) 2.35 +/- 0.11 at 24 degrees C, D(0) 3.42 +/- 0.13 at -80 degrees C compared to D(0) 1.81 +/- 0.01 or 2.53 +/- 0.05, P = 0.0087 and 0.0026, respectively). Thus the MnSOD transgene product protects frozen marrow cells as well as marrow cells irradiated at 24 degrees C.     

Assessment of the cell viability of cultured Perkinsus marinus (Perkinsea), a parasitic protozoan of the Eastern oyster, Crassostrea virginica, using SYBRgreen-propidium iodide double staining and flow cytometry

A flow cytometry (FCM) assay using SYBRgreen and propidium iodide double staining was tested to assess viability and morphological parameters of Perkinsus marinus under different cold- and heat-shock treatments and at different growth phases. P. marinus meront cells, cultivated at 28 degrees C, were incubated in triplicate for 30 min at -80 degrees C, -20 degrees C, 5 degrees C, and 20 degrees C for cold-shock treatments and at 32 degrees C, 36 degrees C, 40 degrees C, 44 degrees C, 48 degrees C, 52 degrees C, and 60 degrees C for heat-shock treatments. A slight and significant decrease in percentage of viable cells (PVC), from 93.6% to 92.7%, was observed at -20 degrees C and the lowest PVC was obtained at -80 degrees C (54.0%). After 30 min of heat shocks at 40 degrees C and 44 degrees C, PVC decreased slightly but significantly compared to cells maintained at 28 degrees C. When cells were heat shocked at 48 degrees C, 52 degrees C, and 60 degrees C heavy mortality occurred and PVC decreased to 33.8%, 8.0%, and 3.4%, respectively. No change in cell complexity and size was noted until cells were heat shocked at >or=44 degrees C. High cell mortality was detected at stationary phase of P. marinus cell culture. Cell viability dropped below 40% in 28-day-old cultures and ranged 11-25% in 38 to 47-day-old cultures. Results suggest that FCM could be a useful tool for determining viability of cultured P. marinus cells.     

Comparison of stem cell viability of bone marrow cryopreserved by two different methods

Two different cryogenic methods were used to study the preservation of murine bone marrow cells. Compared to the classical methods, in which separated mononuclear marrow cells in 10% dimethyl sulfoxide (DMSO) were cryopreserved in liquid nitrogen (-196 degrees C), a modified technique was carried out by cryopreservation of unfractionated marrow cells in a mixed protectant of 5% DMSO and 6% hydroxyethyl starch (HES) at -80 degrees C. Samples that were separately thawed after storage for 1, 4, 8, and 12 weeks were assayed for cell viability and recovery of CFU-GM and CFU-S. No macroscopic clumping of cells was noted either in fractionated or in unfractionated marrow cell cryopreservations. A mild damage, about 25% reduction of stem cells, was found at 1 week and did not deepen further. It seems that the greatest loss of stem cells occurred in the process of cryopreservation itself. Compared to prefreeze values, both a high number of cells that excluded trypan blue (87 +/- 3.4%) and a high recovery of CFU-GM (75 +/- 9.8%) and CFU-S (74 +/- 11.2) were observed in unfractionated marrow samples cryopreserved with the DMSO/HES mixture at -80 degrees C for 3 months and these results were very similar to those obtained from fractionated mononuclear marrow cells cryopreserved at -196 degrees C. The DMSO/HES protectant provides a simplified bone marrow cryopreservation technique that should be favorable to clinical application because of its high stem cell recovery and avoidance of cell-separation manipulation.     

Temperature-induced changes in viability, diphenoloxidase and permeability of Mycobacterium leprae

Among mycobacteria secretion of the enzyme diphenoloxidase has been established as a property of Mycobacterium leprae. The antileprosy drug dapsone (DDS), which completely inhibits the enzyme from plant and mammalian sources, does not readily penetrate intact M. leprae. When the drug is complexed with polylysine, it easily permeates the bacteria and produces 100% inhibition of its diphenoloxidase, suggesting a permeability barrier of the cytoplasmic membrane of M. leprae to dapsone. In this study: (1) when the organisms, purified from fresh tissues of experimentally infected armadillos, were treated with dilute alkali or exposed to warmer temperatures, DDS penetrated the bacteria and inhibited the diphenoloxidase. Washing with trypsin had no effect. Dapsone easily permeated the bacilli, purified from tissues stored at 0 degrees C or at -80 degrees C. (2) Diphenoloxidase of freshly-prepared M. leprae was stimulated when the bacteria were exposed to 50 degrees C for 10 min; at 60 degrees C the activity decreased, and at 100 degrees C the enzyme was completely inactivated. When the enzyme was assayed at temperatures below 37 degrees C, the activity was considerably lower, indicating that M. leprae may not be a psychrophilic organism in this respect. (3) The bacteria exposed to 50 degrees C failed to multiply in mouse footpads. M. leprae remained viable in tissues stored at 0 degrees C or -80 degrees C; but when the bacteria purified from these tissues were frozen, they lost their viability. On the other hand, the organisms separated from fresh tissues remained viable when frozen at -80 degrees C. The inhibition of diphenoloxidase of M. leprae by dapsone could serve as an indirect method to assess the integrity of the bacterial cell membrane and to predict whether the bacteria would retain their viability on freezing.     

Cryopreservation of cultured periosteum: effect of different cryoprotectants and pre-incubation protocols on cell viability and osteogenic potential

Evidence has accumulated that periosteal cells have a great potential to regenerate bone. We have demonstrated that cultured periosteum (CP) in membrane form is an effective device to regenerate alveolar bone. To increase the availability of CP in a clinical environment, an effective cryopreservation protocol for CP has been developed. In this study, three different cryoprotectants (Me(2)SO, glycerol, and ethylene glycol) were used. The effect on cell viability of pre-incubation temperature, pre-incubation time, and agitation during incubation was investigated. Samples were stored at -196 degrees C for 10 days. Cell viability was assessed by a colorimetric cell viability assay using a tetrazolium salt, and the assay results were confirmed by confocal laser scanning microscopy after staining with a combination of calcein AM and ethidium homodimer-1. The activity of the cells after thawing was assessed by alkaline phosphatase assay. To assess the osteogenic potential of cryopreserved CP, the CP was grafted to calvarial defects in athymic rats. The greatest cell viability was obtained in the group equilibrated at 37 degrees C for 30 min with Me(2)SO, under agitation, showing 63.3 +/- 10.5% recovery. After cryopreservation, the cell growth of surviving cells was identical when Me(2)SO was used as a cryoprotectant. Alkaline phosphatase (ALP) activity was maintained in the groups cryopreserved with Me(2)SO and glycerol. The transplantation experiment showed that the calvarial defects were completely closed by grafting cryopreserved CP, which demonstrates that the osteogenic property of CP was well maintained. An efficient cryopreservation protocol for CP has been developed and this will provide a convenient and effective treatment option for bone regeneration in clinics.     

Assay of frozen boar semen with Sephadex filtration

The suitability of filtration of frozen boar semen through Sephadex G-15-120 as a viability assay was investigated. Semen thawed on a hot plate at 38 degrees C was counted with a Coulter Counter before and after filtering through Sephadex columns with 0.1 M sodium citrate as flushing medium. More spermatozoa passed through the column when the temperature of the flushing medium was elevated from room temperature to 37 degrees C and added with 5 mM caffeine (12.3 vs. 22.8% p<0.01). The use of caffeine and 37 degrees C flushings produced a filtrate containing spermatozoa with 89+/-4% motility and 97.6+/-1.6% normal acrosome ridges with the use of frozen semen from 5 different boars. The repeatability was +/-7.3%. The unfiltered samples were judged to contain 45 +/- 8% motile spermatozoa and 66.6 +/- 7.1% spermatozoa with normal acrosome ridges. Filtering of frozen boar semen through Sephadex is proposed as a rapid, objective assay combining the benefits of differential counting of normal acrosome ridges and motility determination.     

Preliminary investigation on the cytotoxicity of tellurite to cultured HeLa cells.

Cytotoxicity of tellurite to cultured HeLa cells was examined by cell viability, lactate dehydrogenase (LDH) assay, and tellurite uptake. The experimental results show that the toxicity of tellurite depends on its concentrations and exposure time. HeLa cells exposed to tellurite for 2 h at 9.1 x 10(-4) to 4.5 x 10(-3) mmol/L did not exhibit cytotoxic effects as measured by cell viability. Exposure to tellurite for 24 h at the same concentrations markedly reduced the cell viability to 57% of the control during the first 5 minutes. Additionally, HeLa cells incubated at 2.7 x 10(-2) to 0.27 mmol/L of tellurite for 2 h retained 53% to 67% of cell viability. Even after 24 h exposure, the HeLa cells incubated at 9.1 x 10(-4) to 4.5 x 10(-2) mmol/L of tellurite still retained 57% to 66% of cell viability. Furthermore, tellurite toxicity was also demonstrated in supernatant of the culture at 37 degrees C by LDH assay. It was found that exposure to tellurite for 90 minutes did not stimulate LDH activity. However, tellurite uptake seems to be more sensitive than the cell viability and LDH activity release tests, as it significantly increases with the increasing of exposure time.     

Role of serum on cryopreservation and subsequent viability of mouse bone marrow hemopoietic stem cells

Normal mouse marrow cells were frozen in an automatically controlled freezer at a cooling rate of 1 °C/min to ?40 °C and 7 °C/ min to ?100 °C using dimethylsulfoxide as a cryoprotective agent. The freezing solution contained in addition either 10% homologous serum or 10% fetal calf serum. Control samples were frozen with serum-free medium. After thawing, stepwise dilution, and washing, the cells were counted, checked for CFU-s content, and cultured in Millipore diffusion chambers for 2 and 7 days.HS resulted in a recovery of 59.7% nucleated cells and 100.5% CFU-s whereas FCS and serum-free medium resulted in 59.8 and 34.7% nucleated cells and 24.5 and 18.2% CFU-s, respectively. After 2 days of culture, D.C. data showed a correlation with the CFU-s results. After 7 days of culture, no significant difference was observed between the three groups. The results of these experiments indicate that HS is required for an optimal stem cell cryopreservation and that a 2-day D.C. culture is a reliable assay system for transplantable hemopoietic tissue.     

Drug metabolism and viability studies in cryopreserved rat hepatocytes

Rat hepatocytes were cryopreserved optimally by freezing them at 1 degrees C/min to -80 degrees C in cryoprotectant medium containing either 20% (v/v) dimethylsulfoxide (Me2SO) and 25% (v/v) fetal calf serum in Leibowitz L15 medium (Me2SO cryoprotectant) or 25% (v/v) vitrification solution (containing Me2SO, acetamide, propylene glycol and polyethylene glycol) in Leibowitz L15 medium (VS25). The VS25 solution was superior for maintaining viability during short-term storage (24-48 hr) but was slightly toxic during longer storage periods (7 days). Although thawed cells were 40-50% viable on ice after cryopreservation, their viability fell rapidly during incubation in suspension at 37 degrees C. This decline in viability occurred more rapidly after freezing in Me2SO cryoprotectant than in VS25 and was associated with extensive intracellular damage and cell swelling. The loss in viability at 37 degrees C does not appear to be due to ice-crystal damage as it occurred in cells stored at -10 degrees C (above the freezing point of the cryoprotectants) and it may be due to temperature/osmotic shock. Both cryoprotectant media were equally efficient at preserving enzyme activities in the hepatocytes over 7 days at -80 degrees C. Cytochrome P450 and reduced glutathione content and the activities of the microsomal enzymes responsible for aminopyrine N-demethylation and epoxide hydrolysis were well maintained over 7 days storage. In contrast, the cytosolic enzymes glutathione-S-transferase and glutathione reductase were markedly labile during cryopreservation. Cytosolic enzymes may be more susceptible to ice-crystal damage, whereas the microsomal membrane may protect the enzymes which are embedded in it.     

Novel cryoprotectant significantly improves the post-thaw recovery and quality of HSC from CB

BACKGROUND: Hematopoietic stem cells (HSC) have traditionally been frozen using the cryoprotectant DMSO in dextran-40, saline or albumin. However, the process of freezing and thawing results in loss of HSC numbers and/or function. METHODS: This study investigated the use of CryoStor for the freezing of HSC from cord blood (CB). CB donations (n = 30) were collected under an Institutional Ethics Committee-approved protocol, volume reduced and frozen using three different methods of cryoprotection. Aliquots were frozen with either 10% DMSO in dextran-40, 10% DMSO in CryoStor or 5% DMSO in CryoStor. Prior to freezing samples were separated for nucleated cell (NC) and CD34+ counts and assessment of CD34+ viability. Aliquots were frozen and kept in vapor phase nitrogen for a minimum of 72 h. Vials were rapidly thawed at 37 degrees C and tested for NC and CD34+ counts and CD34+ viability and colony-forming unit (CFU) assay. RESULTS: Cells frozen with CryoStor in 10% DMSO had significantly improved NC (P < 0.001), CD34+ recovery, viable CD34+ (P < 0.001) and CFU numbers (P < 0.001) compared with dextran in 10% DMSO. CryoStor in 5% DMSO resulted in significantly improved NC (P < 0.001) and CFU (P < 0.001). Discussion: These results suggest that improved HSC recovery, viability and functionality can be obtained using CryoStor with 10% DMSO and that similar if not better numbers can be obtained with 5% DMSO compared with dextran-40 with 10% DMSO.     

Long-term cultures of chicken bone marrow cells

We report an adaptation to cultures of chicken bone marrow cells of the Dexter culture technique for obtaining long-term hemopoiesis in vitro. Cells were seeded in DMEM supplemented with fetal calf serum (20%) and hydrocortisone (10(-6) M) with or without chicken serum (1%). Cultures were incubated at 37 degrees C and fed every 2 weeks. An adherent cell layer composed of macrophages, fibroblasts, and adipocytes became established, over which hemopoietic cells formed foci and were released into the supernatant. Granulocytes and monocytes-macrophages differentiated in a constant proportion until Week 6, whereafter differentiation became progressively restricted to the monocytic lineage. As demonstrated by the generation of colony-forming cells, hemopoiesis was maintained for either 12 or 28 weeks.     

Intracellular ice formation during the freezing of hepatocytes cultured in a double collagen gel.

During freezing, intracellular ice formation (IIF) has been correlated with loss in viability for a wide variety of biological systems. Hence, determination of IIF characteristics is essential in the development of an efficient methodology for cryopreservation. In this study, IIF characteristics of hepatocytes cultured in a collagen matrix were determined using cryomicroscopy. Four factors influenced the IIF behavior of the hepatocytes in the matrix: cooling rate, final cooling temperature, concentration of Me2SO, and time in culture prior to freezing. The maximum cumulative fraction of cells with IIF increased with increasing cooling rate. For cultured cells frozen in Dulbecco's modified Eagle's medium (DMEM), the cooling rate for which 50% of the cells formed ice (B50) was 70 degrees C/min for cells frozen after 1 day in culture and decreased to 15 degrees C/min for cells frozen after 7 days in culture. When cells were frozen in a 0.5 M Me2SO + DMEM solution, the value of B50 decreased from 70 to 50 degrees C/min for cells in culture for 1 day and from 15 to 10 degrees C/min for cells in culture for 7 days. The value of the average temperature for IIF (TIIF) for cultured cells was only slightly depressed by the addition of Me2SO when compared to the IIF behavior of other cell types. The results of this study indicate that the presence of the collagen matrix alters significantly the IIF characteristics of hepatocytes. Thus freezing studies using hepatocytes in suspension are not useful in predicting the freezing behavior of hepatocytes cultured in a collagen matrix. Furthermore, the weak effect of Me2SO on IIF characteristics implies that lower concentrations of Me2SO (0.5 M) may be just as effective in preserving viability. Finally, the value of B50 measured in this study indicates that cooling rates nearly an order of magnitude faster than those previously investigated could be used for cryopreservation of the hepatocytes in a collagen gel.     

Characterization of cells isolated and cultured from human bone

Cells isolated from samples of human iliac crest and human femoral heads by collagenase digestion have been successfully cultured in Fitton-Jackson modified BGJb culture medium supplemented with penicillin (100 units/ml), streptomycin (100 micrograms/ml), and fetal calf serum (10%). Although only a low proportion of the cells survived the initial plating (less than 1%), cells established in culture were readily passaged. Examination of cells obtained at intervals during the collagenase digestion showed that the percentage of cells that attached increased with time of digestion. Rapid sample preparation of rat bone did not substantially increase the number of cells attaching. Thus, it seems unlikely that the low survival was due to loss of viability during sample transportation and preparation. Of several media tested BGJb supplemented with 10% fetal calf serum supported the best growth. Population doubling time averaged 104 hr. Cultured human bone cells were assayed for alkaline phosphatase activity using the azo dye method with naphthol ASTR phosphate as the substrate. A portion of the cells (19%) demonstrated high activity in all cultures examined regardless of the passage number of the culture. Autoradiography of cells exposed to [3H]thymidine showed incorporation of the label into both alkaline phosphate-positive and -negative cells. The stimulation of cell proliferation by growth factors was studied by determining the incorporation of [3H]thymidine into DNA. The specific skeletal growth factor from human bone stimulated cell proliferation several-fold with a half-maximal effect at 5 micrograms/ml. Insulin, epidermal growth factor, and a crude preparation of somatomedin C also stimulated cell proliferation.