Non-frozen Cells Research Articles

Technical note: improving the efficiency of generating bovine extraembryonic endoderm cells.

The formation of extraembryonic endoderm (XEN) occurs early in embryonic development. The cell types that develop from the XEN remain poorly studied in ruminant species because of the lack of suitable cell culture model systems. The goal of this work was to establish a protocol for producing XEN cell cultures from bovine blastocysts. Previous work identified fibroblast growth factor 2 (FGF2) as a facilitator of bovine XEN development. Further refinements in culture conditions studied here included exposure to 20% fetal bovine serum and FGF2 replenishment. These modifications yielded an endoderm outgrowth formation incidence of 81.6% ± 5.5% compared with 33.3% ± 5.5% in bovine serum albumin (BSA)-supplemented controls. These cells resembled XEN when examined morphologically and contained XEN transcripts (GATA binding protein 4 [GATA4] and GATA binding protein 6 [GATA6]) as well as transcripts present in visceral (BCL2 interacting protein 1 [BNIP1] and vascular endothelial growth factor A [VEGFA]) and parietal (C-X-C motif chemokine receptor 4 [CXCR4], thrombomodulin [THBD], and hematopoietically expressed homeobox [HHEX]) XEN. Two XEN cell lines were maintained for prolonged culture. Both lines continued to proliferate for approximately 6 wk before becoming senescent. These cultures maintained an XEN-like state and continued to express GATA4 and GATA6 until senescence. An increase in the abundance of visceral and parietal XEN transcripts was observed with continued culture, suggesting that these cells either undergo spontaneous differentiation or retain the ability to form various XEN cell types. Stocks of cultured cells exposed to a freeze-thaw procedure possessed similar phenotypic and genotypic behaviors as nonfrozen cells. To conclude, a procedure for efficient production of primary bovine XEN cell cultures was developed. This new protocol may assist researchers in exploring this overlooked cell type for its roles in nutrient supply during embryogenesis.

Outpatient Autotransplants for Persons with Multiple Sclerosis

Background Intensive chemotherapy and a haematopoietic cell autotransplant is an effective therapy of multiple sclerosis (MS). This is often done in an inpatient setting using frozen blood cells. Objective Determine if intensive chemotherapy and a haematopoietic cell autotransplant can be safely done in an outpatient setting using refrigerated, non-frozen cells. Methods We developed an autotransplant protocol actionable in an outpatient setting using refrigerated blood cells collected after giving cyclophosphamide, 50 mg/kg/d x 2 d and filgrastim, 10 μg/kg/d. A 2nd identical course of cyclophosphamide was given 9 d later followed by infusion of blood cells stored at 4° C for 1-4 d. The co-primary outcomes were rates of granulocyte and platelet recovery and therapy-related mortality (TRM). Results We treated 517 consecutive subjects. Median age was 47 y (range, 21-73 y). 182 (35.2%) were male. 109 (21.1%) had primary progressive MS, 215 (41.6%), relapsing remitting MS and 193 (37.3%), secondary progressive MS. Median blood cell refrigeration time was 1 d (range, 1-4 d). Median interval to granulocytes >0.5 × 10E+9/L was 8 d (range, 2-12) and to platelets >20 × 10E+9/L, 8 days (range, 1-12). 21 subjects (4%) were hospitalized, predominately for MS flare up (N = 5) and neutropenic fever (N = 5). There was 1 early death from infection. Conclusion Intensive chemotherapy and a haematopoietic cell autotransplant can be safely done in an outpatient setting using refrigerated cells.

Amniotic fluid: Source of valuable mesenchymal stem cells and alternatively used as cryopreserved solution

Mesenchymal stem cells (MSCs), which possess remarkable capabilities, are found in amniotic fluid (AF). The findings of several studies have shown the potential benefits of these cells in applications of regenerative medicine. In clinical applications, an over-period of time is required in a preparation process that makes cell collection become more necessary. Herein, the aim of this study was to preserve and characterize the cell’s properties after cell cryopreservation into an appropriate cryogenic medium. The results illustrated that the highest hAF-MSCs viability was found when the cells were conserved in a solution of 5% DMSO + 10% FBS in AF. However, no statistical differences were identified in a chromosomal aberration of the post-thawed cells when compared to the non-frozen cells. These cells could also maintain their MSC features through the ability to express cell prolific quality, illustrating the typical MSC markers and immune privilege properties of CD44, CD73, CD90 and HLA-ABC. Additionally, post-thawed cells were able to differentiate into chondrogenic lineage by exhibiting chondrogenic related genes (SOX9, AGC, COL2A1) and proteins (transcription factor SOX9 protein (SOX9), cartilage oligomeric matrix protein (COMP) and aggrecan core protein (AGC)), as well as to present sGAGs accumulation. Interestingly, the use of a transmission electron microscope (TEM) uncovered the enrichment of the rough endoplasmic reticulum (rER) that coincided with euchromatin and the prominent nucleolus in the chondrogenic-induced cells that are normally found in the cells of natural cartilage. All in all, this study manifested that AF can be a major consideration and applied for use as a co-mixture of cryogenic medium.

Medium composition for effective slow freezing of embryonic cell lines derived from marine medaka (Oryzias dancena).

This study was conducted to identify optimal medium composition for freezing Oryzias dancena embryonic cell lines. Different freezing media consisting of various concentration of dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), and trehalose were prepared and long-term cultured embryonic cell line was frozen in each freezing medium by conventional slow freezing program for 7days. Through measurement of viability and growth of post-thaw cells frozen in each freezing medium, it was determined that optimal composition of three components was 10% DMSO, 20% FBS, and 0.1M trehalose. The post-thaw cells frozen in optimal freezing medium showed similar morphology and growth rate with non-frozen cells. Next, this condition was applied to two different sets of experiment; (1) freezing of the same cells during expanded period (57days) and (2) freezing of short-term cultured cells from other batches for 7days. The viability of post-thaw cells was significantly low and comparable in set 1 and 2, respectively, when compared with the result of long term-cultured cells frozen in optimal freezing medium for 7days and similar morphology and growth rate with non-frozen counterparts were detected in the post-thaw cells from both sets. In conclusion, this study first reports the optimal medium composition for freezing O. dancena embryonic cells, which can contribute to fish species preservation as well as improvement of cell-based biotechnology by providing stable cell storage.

Cryopreserved mouse pancreatic acinar cells from long-term explant outgrowth cultures maintain their secretory phenotype after thawing

Background/objectivesWe recently reported an explant outgrowth culture method for obtaining functionally competent mouse pancreatic acinar cells for long-term in vitro purposes. The aim of the present study was to explore the possibility of cryostoring these cells without loss of functional differentiation. MethodsAcinar cells prepared by the explant outgrowth method were cryopreserved using a DMSO-based protocol and stored in liquid nitrogen for 4 weeks. The following characteristics were compared in cryopreserved and parallel non-frozen cell preparations: cell viability and recovery, amylase content in viable cells before culture, basal and stimulated amylase release in culture and the ability of the cells to form glandular structures in Matrigel. ResultsImmediate post-thaw viability of the cells was similar to that of freshly isolated cells. Approximately 53% of viable cells frozen were recovered after thawing. Intracellular amylase content was identical in frozen and non-frozen cells. Cryopreserved cells maintained their ability to secrete amylase and to respond to caerulein stimulation in 4-day secondary cultures. They also were observed to form amylase-expressing glandular structures in three-dimensional cultures in Matrigel in a similar manner as non-frozen cells. ConclusionsThis study shows that pancreatic acinar cells can be cryopreserved for long-term storage in liquid nitrogen without dedifferentiation. Successful cryopreservation helps to refine the experimental use of primary acinar cells by enabling their banking for on-demand utilization.

Study design may explain discrepancies in GB virus C effects on interferon-γ and interleukin-2 production and CD38 expression in T lymphocytes

Recently, Baggio-Zappia et al. (2011) reported the ef -fects of GB virus C (GBV-C) on interleukin (IL)-2 and interferon (IFN)-γ in CD4 and CD8 T cells. GBV-C coin -fection was not associated with lower immune activation in human immunodeficiency virus (HIV)-positive indi -viduals as measured by CD38 expression per cell and IL-2 and IFN-γ expressing cells in their study. We agree with Baggio-Zappia et al. (2011) that “several factors, such as differences in the study populations, the stage of HIV in -fection and the time of GBV-C acquisition, may account for the discrepant results reported by different research groups”. However, two major factors may confound inter -pretation of the results reported by Baggio-Zappia et al. and provide the most likely explanation for the discrepant results observed between this study and previous reports (Maidana-Giret et al. 2009, Baggio-Zappia et al. 2011).First, approximately 85% of the subjects studied were on potent combination antiretroviral therapy (cART) and had nondetectable HIV RNA levels in their plasma. cART lowers T cell activation, raises CD4 counts, decreases HIV VL and thus interferes significantly with other fac -tors associated with HIV disease progression (Autran et al. 1997, Carcelain et al. 2001, Lederman 2001). Since the vast majority of subjects in the study of Baggio-Zappia et al. (2011) were effectively treated by cART, it is not surprising that GBV-C did not have an observed effect on the clinical variables studied (CD4, HIV VL etc.).Secondly, Baggio-Zappia et al. (2011) froze the periph -eral blood mononuclear cells (PBMCs) prior to conduct -ing in vitro studies of activation, IL-2 and IFN-γ cytokine production. Freeze-thawing PBMCs can alter detection of both surface marker expression and T cell function (Kvarn -strom et al. 2004, Mallone et al. 2011). By comparison, several groups found a reduction in CD38 on CD4 and/or CD8 T cells in HIV-infected subjects using fresh PBMCs (Bhattarai et al. 2011, Maidana-Giret et al. 2009). In addi -tion, HIV entry (chemokine) receptors CCR5 and CXCR4 are upregulated on CD4 and CD8 T cells following T cell activation (Smith et al. 2002). Three independent groups found reduced levels of CCR5 on CD4 cells using fresh, non-frozen PBMCs and one of these studies also found reduced levels of CXCR4 on CD4 cells (Nattermann et al. 2003, Maidana-Giret et al. 2009, Schwarze-Zander et al. 2010). Finally, IL-2 interactions with GBV-C have been demonstrated in vitro (George et al. 2003, Bhattarai 2011) and in vivo (Stapleton et al. 2009) using fresh, non-frozen cells for flow cytometry studies.Since cART will lower detection of cell surface mark-ers of T cell activation, making detection of a diminu-tion by GBV-C more difficult to detect, and since frozen PBMCs can alter levels of T cell activation markers, we believe that the study of Baggio-Zappia et al. (2011) does not negate the prior studies that used fresh PBMCs to evaluate T cell activation. However, the results illustrate the need for more direct and thorough studies of GBV-C on T cell function in individuals with HIV-infection.

Survival of Mycobacterium tuberculosis during experimental aerosolization and implications for aerosol challenge models

We undertook a series of experiments to investigate factors that contribute to variation in Mycobacterium tuberculosis viability and infectivity, during experimental aerosolization, with an aim to optimize a strategy to enable a more reproducible delivered dose within animal models of tuberculosis. The viability and infectivity of the challenge suspension was determined in relation to aerosolization time, concentration, method of preparation and M. tuberculosis strain. Challenge stocks generated from frozen aliquots of M. tuberculosis were shown to undergo a 1 log(10) CFU ml(-1) decrease in viability during the first 10 min of aerosolization. This correlated with a decrease in surface lung lesions developing in guinea pigs challenged during this time. The phenomenon of decreased viability in vitro was not observed when using freshly grown, nonfrozen cells of M. tuberculosis. The viability of aerosolized bacilli at the point of inhalation relative to the point of aerosolization always remained constant. Based on these findings, we have developed an improved strategy by which to reproducibly deliver aerosol infection doses to individually challenged animals and separately challenged groups of animals. Study of the aerobiological characteristics of micro-organisms is a critical step in the validation of methodology for aerosol infection animal models, particularly where large numbers of animals and nonhuman primates are used.

Enzyme induction in cryopreserved human hepatocyte cultures

Freshly isolated human hepatocytes are considered as the gold standard for in vitro testing of drug candidates. Meanwhile also cryopreserved human hepatocyte suspensions are available. However, a drawback of these cells is the incalculability of attachment to the culture dish. Therefore, we established a technique freezing hepatocytes cultured on a collagen gel. After thawing damaged cells were removed to a certain extent by gentle washing with culture medium prior to adding an upper gel layer. The morphology of the resulting hepatocyte cultures could not be distinguished from that of non-frozen cells. However, basal activities of cytochrome P450 isoforms decreased in cryopreserved compared to non-frozen hepatocytes, as evidenced by analysis of testosterone hydroxylation (OHT) in positions 6β, 16α, 2β and 6α. Nevertheless, enzyme induction factors caused by 24 h incubation with 50 μM rifampicin were similar in cryopreserved and non-frozen hepatocytes. In cryopreserved hepatocytes rifampicin caused an increase in mean values of 6β-OHT formation from 57.2 to 157.7 pmol/well/min (2.8-fold), compared to an increase from 115.8 to 269.1 pmol/well/min (2.3-fold) in non-frozen cells. Similarly, 16α- and 2β-OHT showed induction factors of 2.4- and 2.3-fold in cryopreserved compared to 1.6- and 2.4-fold in non-frozen hepatocytes, respectively. In conclusion, human hepatocytes cryopreserved on collagen gels show a clear induction of CYP3A4 by rifampicin, although the basal activities are reduced compared to non-frozen cells.

Cryopreserved porcine hepatocyte cultures

Cryopreservation of freshly isolated hepatocytes is regarded the standard technique for long term storage of liver cells. Frankly, we were not successful in reproducing viability rates of about 70% of that which have been reported by most authors as results of various freezing protocols for hepatocyte suspensions. In fact, we saw mostly devastating results. We assume that intracellular ice crystal formation as well as osmotic changes during freezing and thawing of liver cells cause hazardous effects, especially on membranes of cells after enzymatic isolation, and, thus, generally result in a severe loss in number and impaired specific hepatocyte functions in subsequent culture. We tried to improve results by freezing cell cultures instead. We allowed hepatocytes to regain a more stable condition prior to storage and placed them in tissue flasks in a uniform configuration, rather than to pack cell suspensions in vials or bags under rather indefinable conditions. Porcine hepatocytes (viability 92±2%) were isolated from slaughterhouse organs and cultured in a double gel (sandwich) configuration. At day 3, cultures were rate controlled frozen (RCF) and stored in a cell bank for three hours (Group A) or 14 days at −80°C (Group B), respectively. Non-frozen cells (NF) and cultures subjected to a linear freezing rate of −10°C/min (LFR, Group C) with 3 h of storage served as controls from identical cell batches. Upon thawing, at day 2 of subsequent culture, fluorescence microscopy studies revealed a survival rate of 75±10% (mean±S.D.) in the RCF groups. Time of storage (3 h, 14 d) did not influence results. Survival in Group C was 10±5%. Cell integrity was measured by LDH-release, which indicated a larger damage of cells in the LFR group, and thereby resembled the morphological findings. Functional parameters, such as albumin synthesis and CYT P 450-activity were comparable to non-frozen liver cells at 48 h after thawing in the RCF groups (A+B), and showed significantly higher levels in these groups as compared to the LFR Group (C). We recommend to freeze hepatocytes in culture in a rate controlled fashion rather than cell suspensions. This way a cell bank of cryopreserved hepatocyte cultures for batch controlled investigations can be easily obtained. This could ameliorate the availability of rare (human) cell material and might also improve the quality of data generated from in vitro experiments in hepatology or pharmacology/toxicology with liver cells from identical sources. It remains to be seen whether this technique might also be of value in hybrid bioartificial liver devices to make these systems become readily available upon clinical demand.

Development of an optimal method for the cryopreservation of hepatocytes and their subsequent monolayer culture

Adult rat hepatocytes, of good viability, were routinely isolated using a versatile biopsy perfusion technique. In order to develop an optimal cryopreservation regimen we investigated a variety of factors including the cooling rate, the constituents of the medium, the cryoprotective agents and the thawing conditions. The preferred freezing medium consisted of Leibovitz L15 culture medium supplemented with 10% (v/v) foetal calf serum, 10% (v/v) tryptose phosphate broth, 20% (v/v) dimethylsulphoxide, 100 μg/ml streptomycin and 100 U/ml penicillin. A freezing rate faster than the generally used 1–2°C/min appeared to be optimal for subsequent hepatocyte attachment and survival in culture. Handling conditions after thawing were important for maximal recovery of cells in culture. To assess the functional integrity of hepatocytes in monolayer culture after cryogenic storage a number of parameters were monitored. Biochemical assays included lactate dehydrogenase leakage, protein synthesis, cellular ATP and ADP content, glutathione (reduced form), cytochrome P-450 content, ethoxycoumarin- O-deethylase activity, glucuronide and sulphate conjugation capacity. Cytochrome P-450 content was maintained at levels comparable with those in freshly prepared cultures for up to 24 hr in culture; ethoxycoumarin- O-deethylase activity was also similar to that of freshly prepared cultures. Changes in various biochemical parameters indicated that cryopreservation resulted in subtle damage to hepatocytes and that even cells that excluded dye did not survive as well as non-frozen cells. When human hepatocytes were subjected to the method developed for rat hepatocytes, they survived the trauma of cryopreservation but fewer cells subsequently attached and spread out to form a monolayer culture. These results indicate that human hepatocytes are more sensitive to freezing under the conditions developed for rat hepatocytes. This may reflect a greater sensitivity of the cells to cryopreservation and/or that the cryogenic conditions were not optimal for human hepatocytes.

Function of lymphocytes and macrophages after cryopreservation by procedures for pancreatic islets: Potential for reducing tissue immunogenicity

The survival of tissue allografts can be extended by pretreating the tissue to remove the stimulatory leucocytes that populate the graft; with this in mind, we have recently begun to explore a cryobiological approach to modulating tissue immunogenicity by using the differential susceptibility of different cells to freezing injury. The sensitivity of leucocytes to fast cooling rates, which were used in procedures that have been reported to yield viable pancreatic islets of Langerhans, was examined. The loss of both cell numbers and the ability of peripheral blood lymphocytes to undergo blastogenic transformation in response to the mitogen concanavalin A after freezing and thawing was determined over a range of cell concentrations using the “curve-shift” method. Lymphocytes frozen at 1 °C/min by a control procedure that was designed to yield maximum survival of lymphocytes showed that although there was a decrease in the number of responding cells, there was no reduction in the ability of the recovered cells to undergo blastogenesis when compared with the response of nonfrozen cells. However, cooling at 1 °C/min in the experimental procedures resulted in both the loss of cells as well as a marked reduction in the ability of recovered cells to incorporate 125I-deoxyuridine into nucleic acid. Cells cooled at either 20 or 75 °C/min by any of the procedures showed total inability to respond to stimulation. Lysozyme is produced continuously by all types of macrophages in culture. The large net increase in total lysozyme content of macrophage cultures is therefore a useful measure of the viability of these accessory cells. Cooling at 1 °C/min by a control, optimized procedure yielded 91% survival of viable peritoneal exudate cells. Cooling at either 1 or 20 °C/min in the experimental procedures resulted in 72–75% survival of cells frozen by one method and 33% survival when frozen by an alternative procedure. Negligible recovery of viable cells was obtained after cooling at 75 °C/min. The preservation protocols employed in this study differ significantly in the variables known to influence the survival of the cells; these include the concentration of cryoprotectant (CPA), the length and temperature of exposure to CPA, the dilution regimen, and the optimum cooling rate for survival of pancreatic islets. This study therefore defines clearly those conditions most likely to effect a depletion of “passenger” lymphoid cells by freezing during the Cryopreservation of islets of Langerhans.

Cryopreservation of isolated rat hepatocytes

Isolated parenchymal hepatocytes from adult rats were frozen in media containing 10% glycerol, 10% dimethylsulfoxide (DMSO), 20% DMSO. Thus microsome-associated functions were compared in nonfrozen cells and cells frozen in each of the above cryoprotectant solutions. Freezing in DMSO maintains cytochromes P-450 and b5 and NADPH-cytochrome C reductase at levels nearer to control values than does freezing in glycerol. Cells frozen and subsequently thawed and cultured for 24 h lose a greater amount of cytochrome P-450 than do nonfrozen cultured cells. The levels of cytochrome b5 and reductase in frozen-thawed cells remain close to control values. Cell viability (trypan blue dye exclusion and percentage of attached cells) after freezing is maintained better using DMSO as a cryoprotectant. Dimethylsulfoxide protects the hepatocytes from freeze-induced damage to the extent that many viable cells attach to collagen-coated petri dishes, survive for at least 24 h, and still maintain significant levels of enzymes of importance to drug and carcinogen metabolism.

Calculation of protoplast freezing strain in multicellular plant tissues

A method for calculating the contraction strain (or the converse stress) to protoplasts of frozen multicellular plant tissues is described. The method requires (i) a nuclear magnetic resonance (NMR) measure of the quantity of bound water per gram dry weight ( K), (ii) a gravimetric measure of grams of H 2O per gram dry weight ( L 0), (iii) a measure of solute concentration in non-frozen cells ( C 0), (iv) an estimate of the specific volume of tissue dry matter (0.625 ml/g), (v) an NMR measure of the fraction of tissue water that is intracellular osmotic water ( P a), and (vi) a measure of the fraction of the dry weight that is cell wall ( f cw). This method is a refinement of previous methods that calculate cell contraction strain from four ( L 0, K, C 0, and 0.625) of the above six measurements. Comparison of the calculated protoplast strain to the calculated cell strain indicates that the two measures are quite similar, however, the measure of protoplast strain is, in theory, a more appropriate measure of the freezing strain. It is also demonstrated that derivation of a measure of strain from the parameters controlling it is useful, because it allows one to evaluate the relative contribution of each parameter in preventing the development of strain.

Large-scale purification and cryopreservation of human monocytes

Human blood monocytes were purified by a new method capable of handling 3 × 10 9 mononuclear leukocytes, which does not involve adherence of the cells and takes about 3 h to perform. The yield of monocytes is 70%, the purity about 90% and the viability 99%. Monocytes purified by this method were cryopreserved at −196°C. The function of the cells was tested before freezing and after thawing. We found that the capacity of cryo-preserved monocytes to move to the source of a chemotactic gradient, to ingest particles, to mount a respiratory burst during phagocytosis, to kill intracellular bacteria, to lyse anti- d-sensitized erythrocytes and to help autologous lymphocytes in a proliferative response to mitogens or antigens, was preserved by 70% or more as compared with non-frozen cells. Thus, cryopreservation of human blood monocytes is possible with maintenance of functional capacities.

Binding of Concanavalin A to Chinese Hamster Ovary cells after storage in liquid nitrogen

The binding of Concanavalin A to the surface of Chinese Hamster Ovary (CHO) cells after storage in liquid nitrogen was studied by a number of techniques. Electron microscope cytochemistry revealed that cells frozen at 1.7 °C/min resulting in high viability had a Con A positive surface coat similar to non-frozen control cells whereas those that had been frozen at 200 °C/min, resulting in low viability, exhibited a range of staining patterns with many cells showing a loss of the coat. However, the binding of I 125-labeled Con A to the frozen cells revealed no overall loss of binding sites as compared to non-frozen cells although those frozen at 1.7 °C/min appeared to have two types of binding sites whereas the cells frozen at 200 °C/min had only one. Agglutination studies showed that the frozen cells had reduced levels of agglutinability.

Comparing stimulation of lymphocytes in different samples: Separate effects of numbers of responding cells and their capacity to respond

A range of concentrations in culture of human lymphocytes from the peripheral blood has been used to provide information on two major factors contributing to the total response to stimulation with mitogens; these factors are the numbers of responding cells, and the efficiency of these responding cells. The marked effect of cell concentration in culture on the thymidine incorporation of stimulated lymphocytes ranged from a background response to a maximal response mainly over 1 log unit of cell concentration (cells/ml). At higher cell concentrations the measured response fell. Since the absolute number of lymphocytes that respond to stimulation was not known, the relative positions of these responses (incorporations of thymidine) on the axis of concentration of cells were used to estimate the relative numbers of responding cells in different samples. The efficiency of responding cells in different samples was compared using the shape of the response curve with changing concentration of cells. With this approach, frozen and thawed samples of lymphocytes were compared with non-frozen cells and the results indicated a loss in the numbers of responding cells after freezing and thawing, but no change in their efficiency of incorporating thymidine on stimulation with phytohaemagglutinin or allogeneic cells.

Cryopreservation of flagellar mutants of Chlamydomonas reinhardtii

Ten strains of Chlamydomonas reinhardtii flagellar mutants were successfully cryopreserved using two cryogenic freezing methods. Dimethylsulfoxide (DMSO) was found to be the only effective cryoprotectant of those tested. After recovery, cells were viable and morphologically indistinguishable from non-frozen cells. No recovery was obtained after lyophilization.

An ultrastructural study of the recovery of Chinese hamster ovary cells after freezing and thawing.

The effect of freezing on the recovery of Chinese hamster tissue cells has been studied by freezing cells at a rate known to give high recovery and comparing these under the electron microscope with nonfrozen trypsinized cells for periods up to 14 hr after treatment. The main areas of damage were the cell surface and the cytoskeletal framework of the cell. The microfilament and microtubule systems underlying the cell membrane were shown to be disrupted in both the frozen and nonfrozen cells but repolymerization and reorganization was shown to be retarded for a longer period in the frozen cells. A greater degree of surface blebbing was observed in the frozen cells and heterochromatin was densely stained. The delay in return of the frozen cell to a normal morphology and physiology may be due to the need for the cell to repair sublethal cell damage before normal physiological processes can continue.

Improved assay for monocyte chemotaxis using frozen stored responder cells

Frozen stored human mononuclear cells were compared with non-frozen cells as responder cells in the monocyte chemotaxis assay. Frozen stored cells were found to exhibit lower background migration, resulting in greater experimental-to-control ratios. The use of frozen cells also greatly improved the speed, standardization of conditions and day-to-day reproducibility of the assay procedure. Frozen cells were at least as sensitive as fresh cells for detecting low chemotactic factor concentrations and were superior for detecting differences in concentration.

DNA profile of the spore of Blastocladiella emersonii: evidence for -particle DNA.

Procedures were developed for isolating DNA from zoospores of Blastocladiella emersonii, critical steps being the use of non-frozen cells, enzymatic elimination of polysaccharide, and a combined treatment with T1RNAse and pancreatic RNAse for complete removal of RNA. Methods for isolating γ-particles from spores were also established. Extraction of DNA directly from purified γ-particles substantiated previous assumptions that they contain DNA. Heat denaturation and preparative CsCl equilibrium sedimentation studies of whole spore DNA revealed the presence of four distinct components, I–IV, with buoyant densities of 1.731, 1.715, 1.705, and 1.687 g/cm3. The major species appeared to be nuclear chromatin. Nucleolar and mitochondrial sites were tentatively assigned to species II and III, respectively. Evidence was presented that species IV resided in γ-particles. Some possible roles for γ-particle DNA were discussed.