Effects of cell concentration on viability and metabolic activity during cryopreservation.

Increasing the cell concentration during the cryopreservation of red blood cells (RBC) increased hemolysis. Similarly, increasing the cell concentration during the cryopreservation of hepatocytes reduced both the viability of the cells as assessed by trypan blue exclusion and the metabolic activity of the trypan blue-excluding cells. In both cell types, significant damage appeared at cell concentration levels exceeding 60%. With tightly packed RBC, hemolysis reached 63%, while for hepatocytes after thawing and dilution, trypan blue viability was reduced to 41% of that of the cells initially isolated. The viability of cryopreserved hepatocytes, estimated by trypan blue exclusion, was considerably reduced following incubation for 1 h at 37 degrees C and the extent of this reduction was also a function of cell concentration during freezing and thawing. The trypan blue viability of hepatocytes that were tightly packed during cryopreservation and then incubated at a concentration of 5 x 10(6) viable cells/ml fell dramatically to only 12.5%. The protein-synthesizing activity and the membrane transport activity of these cells, expressed in terms of cells that excluded trypan blue immediately following thawing and removal of the cryoprotectant, were reduced to 38.7 and 33.5%, respectively, of the activity in cells that were packed at 10% cell concentration. The fall in metabolic activity may have been due to complete loss of activity in some of the cells or reduced activity in most or all of the cells or any combination of these factors. It is concluded that there may be many mechanisms involved, but the most important factor is probably stresses produced by unphysiological cell-cell contacts occurring during the freeze-thaw cycle.

Cryopreservation of isolated rat hepatocytes: effects of iron-mediated oxidative stress of metabolic activity.

In an attempt to quantitatively evaluate the destructive effects of free radicals on metabolism, freshly prepared and cryopreserved isolated rat hepatocytes were exposed to and incubated with Fe2+ compounds, reputedly inducing oxygen-derived free radicals (OFR) capable of attacking the lipid structures of cellular membranes. Malondialdehyde (MDA) formation was interpreted as an expression of free radical interaction with polyunsaturated lipids, and in vitro incubations were carried out during the period of constant MDA formation. Protein synthesizing activity was evaluated by incubating control hepatocytes and cells previously exposed to 100 microM of Fe2+, to 100 microM of Fe2+, and 100 microM of desferrioxamine and to 100 microM of desferrioxamine alone with 0.1 microCi of L-[U-14C]isoleucine and in the presence of these compounds. Membrane transport activity was similarly evaluated by following the cellular uptake of alpha-amino-[1-14C]isobutyric acid. Protein-synthesizing activity of freshly prepared and cryopreserved hepatocytes was not affected by Fe2+ treatment, nor by the additions of the iron chelator desferrioxamine. Amino acid transport, however, was inhibited by 100 microM of Fe2+, but was effectively neutralized by the simultaneous addition of 100 microM of desferrioxamine. Cryopreserved hepatocytes equally presented a significantly inhibited amino acid transport activity over the incubation period. The results suggest that the metabolic depression measured in thawed hepatocytes does not result to any large extent from iron-catalysed OFR effects. When OFR production was deliberately induced, the most significant early change was seen in transmembrane amino acid uptake in both fresh and cryopreserved cells.

Osmotic effects of dilution on erythrocytes after freezing and thawing in glycerol-containing buffer.

Red blood cells frozen in 1.7 M and particularly in 2.2 M of glycerol retain a high degree of integrity upon thawing as long as the dilution procedure of the cryoprotectant is slow and preferentially compensated by the addition of sorbitol. As the nonpenetrating cryoprotectant sorbitol induces initial cell shrinkage, cell swelling upon dilution of the cryoprotectants may not lead to hemolysis. However, rapid dilution of glycerol even with buffer containing up to 0.50 M of sorbitol cannot be achieved without provoking considerable hemolysis. Due to the relative slow rate at which glycerol leaves the cells, membrane damage to the younger cell populations remains considerable and is even more pronounced in the older cell groups. The dramatic osmotic changes occurring during the dilution process lead to the formation of aberrant cell populations as demonstrated by the red cell size frequency distribution curves.

Metabolic activity of freshly prepared and cryopreserved hepatocytes in monolayer culture.

The successful use of isolated hepatocytes for transplantation will, no doubt, require cryopreservation of the cells. However, cryopreservation results in the loss of viability of isolated hepatocytes. In this study a method is described that allows recovery of viable hepatocytes after cryopreservation. Freshly prepared and cryopreserved hepatocytes (1.8 M Me2SO in Euro-Collins solution) were suspended in Dulbecco's culture medium and allowed to form monolayers in Primaria T-25 culture flasks. Viable hepatocytes produced monolayers after 3 h of incubation and viable cultures could be maintained for up to 7 days as judged by trypan blue exclusion. Metabolic viability was measured by incorporation of radiolabeled isoleucine into proteins. Maximal protein synthetic capabilities in freshly prepared hepatocytes was obtained 24 h after culturing. Cryopreserved hepatocytes showed a decrease in protein synthetic capabilities after 24 h culturing. However, longer times of culture resulted in regenerating the capacity of these cryopreserved cells to carry out protein synthesis to levels similar to those of freshly prepared and cultured cells. Thus, incubation of monolayer cultures of cryopreserved hepatocytes for at least 48 h provides a means for reversing the injury caused by freeze-thaw stress and the regeneration of initial viability. This technique may provide a method that is suited for the use of cryopreserved hepatocytes for clinical transplantation.

Effects of sodium ascorbate (vitamin C) and 2-methyl-1,4-naphthoquinone (vitamin K3) treatment on human tumor cell growth in vitro. II. Synergism with combined chemotherapy action.

The growth inhibitory effects of a combined application of sodium ascorbate (Vitamin C) and 2-methyl-1,4-naphthoquinone (Vitamin K3) together with various chemotherapeutic agents has been examined on in vitro cultured human endometrial adenocarcinoma (AN3CA) cells. Combined vitamin treatment and chemotherapy in well defined conditions of cell confluence and at the dose levels applied result in a synergistic effect on growth inhibition. The combined vitamins when reaching their own synergistic cytotoxicity levels frequently obscure the additional synergistic effects attributable to the chemotherapeutic agents. Apart from the specific cytotoxic characteristics of the chemotherapeutic drugs examined, the formation of reactive oxygen radicals during treatment, possibly accentuated by less defined secondary mechanisms, appears essentially responsible for the observed stimulated cytotoxicity.

The effects of cryopreservation on protein synthesis and membrane transport in isolated rat liver mitochondria.

Protein synthesizing activity and membrane transport were examined in fresh and cryopreserved isolated rat liver mitochondria. In the presence of 0.6, 1.2, and 1.8 M final concentrations of dimethyl sulfoxide (Me2SO), both metabolic parameters were considerably inhibited in the fresh samples and even more inhibited in the cryopreserved specimens. However, simple exposure to this penetrating cryoprotectant, followed by its subsequent removal by washing, did not seem to affect significantly the examined functions. When different freeze-thaw regimes were investigated, it was observed that optimal recovery of protein synthesis and membrane transport functions were obtained when fast freezing took place in the absence of Me2SO.

The effects of cryopreservation on membrane integrity, membrane transport, and protein synthesis in rat hepatocytes.

The cryopreservation of hepatocytes is of particular interest as a step in the possible treatment of some inborn disorders of metabolism. This study examines the metabolic damage that occurs as a result of the freeze-thaw procedures and during subsequent incubation periods of isolated rat hepatocytes. Even for freshly prepared hepatocytes, the presence of 1.8 M of Me2SO during incubation led to a rapid decline in viability. Optimal recovery after cryopreservation was obtained when incubation was started after the progressive removal of Me2SO. A buffer medium characterized by an intracellular electrolyte composition (Euro-Collins) proved particularly beneficial to the membrane integrity, probably by protecting the (Na+,K+)ATPase pump activity. The interpretation of viability using the trypan blue exclusion test was generally confirmed by the metabolic analysis of protein synthesizing activity and membrane transport function which are regarded as more rigorous tests of functional viability. The incorporation of L-[U-14C]isoleucine into the proteins of fresh hepatocytes during the first hour of incubation progressively leveled off over the next 2 hr. The cryopreserved hepatocytes showed a similar pattern although at a lower level of activity. Even after 3 hr of preincubation, the subsequent addition of labeled isoleucine still indicated a residual protein synthesizing activity. The active transport of alpha-amino[1-14C]isobutyric acid through the cell membranes reached a peak value after 60 min of incubation of fresh hepatocytes, and after 40 min of incubation of cryopreserved cells, followed by a steep decline as expression of rapid membrane deterioration. Again, the membrane transport pattern for the cryopreserved samples occurred at a lower level of activity. After preincubation of fresh and cryopreserved hepatocytes for 180 min, subsequent addition of labeled alpha-aminoisobutyric acid did not show any further significant metabolic activity. Initially the amino acid availability appeared to control protein synthesizing activity while, as membrane transport became seriously damaged, incorporation leveled off with only a low metabolic activity remaining. Although cryopreserved hepatocytes were susceptible to faster deterioration during subsequent incubation, considerable metabolic activity was retained. However, fresh and cryopreserved hepatocytes expressed metabolic functions at significantly different activities. Moreover, the differences between fresh and cryopreserved cells varied with the particular cellular function being examined.

The bioenergetics of mitochondria after cryopreservation.

The functional characteristics of rat liver mitochondria after cryopreservation with and without the addition of the cryoprotectant dimethyl sulfoxide (Me2SO) were evaluated. As criteria of functional integrity, polarographic measurements of substrate-linked oxygen consumption and luminescent assay of adenosine triphosphate (ATP) synthesis were considered before and after cryopreservation. The results demonstrated that mitochondrial damage after freezing was indicated by the polarographic studies but was not evident when ATP synthesis was considered. Me2SO present during cryopreservation was partially protective for mitochondrial substrate-linked oxygen consumption; however, simple exposure to and dilution from Me2SO effected some changes in mitochondrial function.

Effects of pulsed electromagnetic fields on rat skin metabolism.

In an attempt to approach the mechanism of action of pulsed electromagnetic fields (PEMF) on biological systems, the effects on protein synthesizing activity and on membrane transport have been examined in rat skin. PEMF characterized by specific physical parameters stimulate the incorporation of L-[U-14C]isoleucine into the proteins of rat skin as well as the alpha-amino[1-14C]isobutyric acid uptake during incubation in buffer medium with extracellular electrolyte composition. Analogous incubation experiments carried out in an intracellular medium results in an inhibitory effect of PEMF on both biological functions. Addition of 10(-3) M ouabain to the incubation medium, partially blocking the Na+/K+-ATPase pump mechanism, apart from reducing amino acid transport, results in an overall disappearance of any stimulatory effects by PEMF. PEMF applied to the skin in the presence of 10(-3) M 2,4-dinitrophenol uncoupling the oxidative phosphorylation in the mitochondria and seriously restricting protein synthesis, still provides a limited stimulatory effect on protein synthesizing activity and on membrane transport. The effects of PEMF may well be understood by an increased availability of precursor elements controlled at the cell membrane level. Indeed the observed effects may even be simulated outside electromagnetic fields by modifications in the electrolyte composition of the incubation medium.

Effects of sodium ascorbate (vitamin C) and 2-methyl-1,4-naphthoquinone (vitamin K3) treatment on human tumor cell growth in vitro. I. Synergism of combined vitamin C and K3 action.

The effects of sodium ascorbate (vitamin C) and 2-methyl-1,4-naphthoquinone (vitamin K3) administered separately or in combination on the in vitro cultured human neoplastic cell lines MCF-7 (breast carcinoma), KB (oral epidermoid carcinoma), and AN3-CA (endometrial adenocarcinoma) have been examined. When given separately, vitamin C or K3 had a growth inhibiting action only at high concentrations (5.10(3) mumol/1 and 10(5) nmol/l, respectively). Combined administration of both vitamins demonstrated a synergistic inhibition of cell growth at 10 to 50 times lower concentrations. At this level separately given vitamins are not toxic. The sensitivity to this treatment was somewhat different in the three cell lines, being slightly higher for KB line. This tumor cell growth inhibitory effect was completely suppressed by the addition of catalase to the culture medium containing vitamins C and K3, suggesting an excessive production of hydrogen peroxide as being implied in mechanisms responsible for the above-mentioned effects.

Cytotoxic effects of müllerian inhibiting substance (MIS)?

To determine whether Müllerian Inhibiting Substance (MIS) is the factor responsible for the cytotoxic effects of testicular preparations on gynecological cancers, homogenates of bovine fetal testes were fractionated by Sephadex G 200 chromatography and their cytotoxic effects tested on a Müllerian-derived endometrial cancer cell line (AN3-CA), an ovarian carcinoma cell line (CAOV 3) and on a breast cancer cell line (MCF 7). No difference in cytotoxicity was found between Müllerian and non-Müllerian derived human cancer cells. Cytotoxicity and MIS activity were found in different fractions on Sephadex G 200 gel chromatography.

Influence of practice on the biochemical analysis of steroid receptors in human breast cancer.

The value of experience and practice in the routine biochemical analysis of steroid receptors was studied in 2576 different primary breast cancer specimens over five consecutive years. The positivity rate (beyond 3 fm/protein) and the measured concentrations of the steroid receptors increase. The positivity rate and average steroid receptor concentration of the samples immediately frozen is significantly higher compared to the overall sample population. It is concluded that the handling of the specimen before freezing and the experience of the technician play an important role in the routine biochemical analysis of steroid receptors.

Preservation of taenia coli by freezing and storage at -196 degrees C.

Some damaging effects that occur during cryopreservation by freezing to -196 degrees C have been evaluated in rabbit taenia coli by analyzing the proportional recovery of acetylcholine- and histamine-induced maximal contractions. Dimethyl sulfoxide (Me2SO) 10 v/v% was used as the cryoprotectant; it reversibly abolishes spontaneous contractility even after incubation at 37 degrees C during 2 hr. Programmed freezing at 0.6 degrees C/min with compensation for the latent heat of fusion and warming at 35 degrees C/min proved to be slightly superior to programmed cooling without compensation and slower warming. The degree of functional recovery was comparable after either abrupt or stepwise removal of Me2SO. Freeze-thawing resulted in a significant reduction of contractile force in each buffer solution tested, and acetylcholine-induced contractility was always better preserved than histamine-induced contractility. The best preservation (approximately 65%) was obtained in a potassium-rich buffer solution. The absence of calcium and magnesium from the incubating medium had no influence, whereas the presence of EDTA significantly affected functional recovery. It is difficult to compare our results with those reported by others because of multiple methodological differences. However, it seems that previous results can be improved by changing the freezing rate and the composition of the incubating and cryoprotecting medium.

Ca2+ transport and permeability in inside-out red cell membrane vesicles after freezing.

To evaluate the effects of freezing and thawing on Ca2+ transport and permeability, inside-out red cell membrane vesicles (IORCMV) are examined. Exposure to the cryoprotectant Me2SO as well as different cooling regimes on unprotected and cryoprotected vesicles do not affect the membrane Ca2+ transport. However, freezing and thawing increase the membrane permeability to sucrose.

Biochemical and histochemical analysis of steroid hormone binding sites in human primary breast cancer.

Mammary carcinoma tissue from 514 primary breast cancer patients were all biochemically and histochemically analyzed for both estrogen receptors and progesterone receptors. The dextran-coated charcoal (DCC) method measured the ER and PR as defined by Scatchard analysis, ligand competition experiments and target organ specificity. The ligands, estradiol-6-carboxymethyloxime-BSA-fluoresceine isothiocyanate and hydroxyprogesteronehemisuccinate-BSA-tetramethylrhodamine isothiocyanate, used for histochemistry, did not bind to either ER or PR and were mainly bound to the membrane fraction of isolated breast cancer cells. Fluorescence was not specifically inhibited by estrogens or progestogens. In addition, "estrogenic" always coincided with "progestogenic" fluorescence. The binding of the fluoresceine compounds to tissue slides depended on the large steroid hormone substitution on the bovine serum albumin molecule. Clinical parameters, known to be related to ER and PR did not correlate with the histochemical results. The observations indicated the impossibility of specific steroid receptor detection by the histochemical method. Therefore, up to the present, evaluation of hormone dependency and prognosis in human breast cancer cannot be based on this approach.

Interaction of ionizing irradiation with steroid receptors in human breast cancer cells.

Human mammary tumor cells in continuous culture (MCF-7 cells) are hormone- and radiosensitive. The interaction of both factors is analyzed. Ionizing irradiation lowers the concentration of both the estradiol and progesterone receptors per cell. The reduction is dose dependent. However, the effects on the cytoplasmic and nuclear forms of the receptors are not similar. For the estradiol receptor, an accumulation in the nuclear fraction is observed 48 hr after irradiation when no appreciable amounts of estrogens are present. After administration of 10(-8) M estradiol, the cytoplasmic clearance is comparable to the unirradiated controls. However, nuclear accumulation is impaired. The processing of the nuclear estrogen receptor remains identical. Nuclear progesterone receptor is not significantly increased due to irradiation in the absence of progestins. Cytoplasmic decrease after incubation with progestins is unaffected. Again, nuclear accumulation is impaired in contrast to the unchanged processing of the nuclear form of the progesterone receptor. A decrease in "nuclear acceptor sites" for both receptors after irradiation may be an explanation for these observations. No significant effects of ionizing irradiation are observed in the initial steps of steroid hormone action.

The interaction between different phorbol derivatives and cortisol.

Phorbol derivatives (phorbol-12,13-diacetate, phorbol-12,13-dibenzoate, phorbol-12,13-dibutyrate, and phorbol-12-myristate-13-acetate) interact with cortisol on a molecular basis. These molecular interactions are demonstrated via dexamethasone receptor assays and by changes in spectrophotometric characteristics when equimolar solutions of these phorbol compounds together with cortisol are compared to those of the pure solutions. The phorbol compounds characterized by modifications in the molecular ring structure and by substitution at position C20, lose the capacity to bind cortisol. The tumor promoting effects of phorbol compounds are apparently achieved, in addition to other well-known mechanisms, by neutralizing cortisol, thus suppressing its regulatory effect on cell proliferation.

Effects of daily anti-estrogen treatment on uterine growth and progesterone receptor concentrations in adult rat uterus.

The effects of daily Tamoxifen treatment on adult rat uterus weight depend on the degree of estrogenic impregnation of the animal. In intact animals, Tamoxifen caused a decrease of the uterine weight. A weight increase was observed, after castration, which was dose-dependent without reaching saturation even with 500 micrograms of Tamoxifen per day. This uterine weight increase, reaching its peak during the first 24 h after the initial Tamoxifen application, is attributable to water retention. The uterine progesterone receptor (PgR) concentrations are stimulated by daily antiestrogen treatment of intact and castrated animals. This increase in the PgR levels is initially dose-dependent up to 10 micrograms of Tamoxifen per day reaching maximal levels after 4 days of treatment. The results indicate that growth and PgR induction in adult rat uterus are essentially controlled by independent mechanisms, constituting the biological basis of the synergistic effects of anti-estrogens and progestagens.

Erythrocyte swelling after rapid dilution of cryoprotectants and its prevention.

These in vitro studies on canine red blood cells confirm that cell swelling occurs after rapid dilution of Me2SO and glycerol. Cells loaded with a penetrating cryoprotectant in a medium with low Na+, high K+ composition present significantly less swelling after rapid dilution of the cryoprotectant than cells exposed to an electrolyte medium characterized by high Na+, low K+ composition. The osmotic cell stress during rapid dilution of Me2SO can be completely counteracted by the simultaneous use of the nonpenetrating sorbitol during exposure and loading. However, the addition of sorbitol is of no important benefit when glycerol is used as the intracellular cryoprotectant. This is probably due to the slower elution of glycerol. Thus utilizing a perfusion solution containing sorbitol during loading and dilution of Me2SO reduces the osmotic injury and may greatly improve the survival prospects of cryopreserved organs by avoiding "out-flow" block.

Independent regulation of growth and steroid receptors in uterus and mammary tumors of rats.

Anti-estrogens and progestagens are synergistically active in the treatment of hormone dependent tumors. The combined action of both compounds in daily treatment schedules are analyzed in rat uterus and in DMBA- induced rat mammary tumors. Tamoxifen in contrast to estradiol does not significantly affect tissue growth, while PgR induction is considerably stimulated by Tamoxifen. It is suggested that the "estrogenic effects" of Tamoxifen and estradiol are separately modulated. When given in sequential combination with anti-estrogens, the anti-tumor response of progestagens is enhanced. Combinations of hormonal treatment based on careful analysis of the regulation processes at target cell level may greatly improve results of anti-tumor therapy.