Postfreeze viability of human renal epithelial carcinoma cells related to phase transformations in ternary solutions

The postfreeze viability of human renal epithelial carcinoma cells frozen in solutions based on a complex physiologic support medium to which additions of NaCl and a cryoprotective agent, either glycerol or dimethyl sulfoxide (DMSO) were made, have been determined by a dye exclusion technique. The support medium consisted of either Eagle's Minimum Essential Medium with Hanks' salts added (MEM) or this same medium supplemented with 20 vol% heat-inactivated fetal calf serum (MEM + FCS). Glycerol was found to be an ineffective cryoprotective agent for these cells, while DMSO was highly effective. Addition of NaCl along with the DMSO further improved the viability of cells frozen at −196 °C. Freezing and thawing rates were found to be important with a slow freezing rate, 2.5 °C/min, and a rapid thawing rate, 240°C/min, yielding the best results.Maximum viability occurred in solutions containing 80 to 95 wt.% (MEM + FCS) with the balance being DMSO and NaCl in the weight ratio of 9:1. In addition to primary ice formation, two nonequilibrium glassy phases were observed during DTA studies of these solutions (10). The exintence of these vitreous states reduces the chances thet cells will be exposed to hypertonic concentrations of salt in the extracellur fluids during freezing-out of primary ice.     

Effect of cryoprotectants on release of various enzymes from buck spermatozoa during freezing

Semen samples from 12 bucks Were extended with 10 different extenders containing glycerol, DMSO, glycerol + DMSO, and glycerol + lactose in varying concentrations as cryoprotective agents. The activities of acrosin, hyaluronidase, alkaline phosphatase (AKP), aspartate aminotransferase (AST), alanine amino transferase (ALT) and lactic dehydrogenase (LDH) were assayed in equilibrated (Prefreeze) and frozen thawed (Postfreeze) semen samples. Significantly (P < 0.01) higher intracellular activity of acrosin was recorded in semen samples extended with lactose than with the other extenders, with the maximum being with Tris yolk glycerol lactose (TYGL(180)). Effects of extenders on acrosin activity were significant (P < 0.01) at both of the pre-and postfreeze stages. However, extracellular activities of hyaluronidase, alkaline phosphatase, transaminases (AST and ALT), and lactic dehydrogenase were significantly higher in extenders containing DMSO than lactose. Leakage of these enzymes was found to increase from the prefreeze to the post freeze stage.     

Role of media, protein and energy supplements on maintenance of morphology and DNA-synthesis of small preantral domestic cat follicles during short-term culture

Small preantral follicles (40 to 90 microns in diameter) from domestic cats were cultured for 10 d using different media (M199 and Dulbecco's MEM) and protein (FCS and BSA) supplements. Culture efficacy was determined by Hoechst 33258 staining and estimation of Brom-desoxyuridine (BrdU)-incorporation into oocytes and granulosa cells. Culture in M199 + FCS and in DMEM + FCS resulted in 21.6% and 38.1%, respectively, of morphologically intact preantral follicles. Adding BSA increased the rate of normal follicles to 51.7% in M199 and to 58.6% in DMEM. Oocytes were found in 40% of the follicles, when DMEM and/or BSA supplementation was used, while M199 with FCS induced acute loss of oocytes in 85% of the follicles. About 10% of the oocytes contained degenerating chromatin. Measurement of BrdU-incorporation during culture allows for quick and effective assessment of follicle viability in vitro. Comparison of M199 and Dulbecco's MEM, both with FCS or BSA and DMEM with or without pyruvate/lactate, indicated that Dulbecco's MEM + BSA without pyruvate and lactate is the best medium for culture of cat follicles. However, further research of suitable medium supplements is needed.     

Manipulation of medium conditions and differentiation in the rat myogenic cell line L6

Myoblasts of the L6 rat cell line were grown in Ham's F12 nutrient medium containing 10% fetal calf serum (F12 + FCS). Although the cells were confluent by 6 days in culture, fusion was not observed even if cultures were maintained for 10–14 days. At least 80% of the cells in such confluent unfused cultures were in the G₁ phase of the cell cycle and less than 5% of the cells in confluent cultures synthesized DNA during a 4-day period. The synthesis of muscle-specific proteins (α-actin, β-tropomyosin, and myosin light chains LC1_emb and LC2_F) was negligible when compared to fused cultures of L6 cells grown for a similar time in Dulbecco's medium with 10% FCS (DME + FCS). When the unfused cultures were shifted from F12 + FCS to DME + FCS, DNA synthesis could be demonstrated in more than 95% of the cells and fusion occurred, indicating that neither proliferative nor myogenic capacity had been irreversibly lost. Raising the levels of calcium, varying the serum concentration from 0 to 20%, or the addition of medium components (present in DME but reduced or absent in F12) all failed to induce fusion in the L6 cells grown in F12. However, L6 cells will fuse in mixtures of F12 + FCS and DME + FCS. Fusion will also occur if L6 cells are grown at clonal density in F12 + FCS supplemented with calcium. While it has not been possible to determine why F12 + FCS is nonpermissive for L6 cells in confluent mass cultures, the results demonstrate that prolonged residence in the G₁ phase of the cell cycle is not a sufficient condition for L6 myoblast differentiation to occur.     

Frozen fetal and neonatal mouse cardiac reimplants for assessing cryoprotective agents

The electrical activity of transplanted syngeneic 17–19 day fetal and 1–9 day neonatal mouse hearts has been studied after freezing to −196 °C in the presence of different cryoprotective agents. Histological examinations were performed after electrical activity (QRS) had been studied for periods in excess of 30 days. EG, DMSO, and glycerol appear equally protective provided that glycerol is added at 37 °C. Methanol, although nontoxic at those concentrations which will protect tissue culture cells, does not offer protection to this type of organized tissue. None of the high molecular weight compounds tested offered cryoprotection in this system. Diffusion of both protective agents and nutrients has been shown to be limiting factors in the survival of frozen-thawed neonatal hearts between 6 and 9 days of age.     

Survival of hippocampal and cortical neurons in a mixture of MEM+ and B27-supplemented neurobasal medium

Serum-free B-27 supplemented neurobasal (NB) and a 10% fetal bovine serum-supplemented Eagle's minimum essential medium (MEM+) are used to culture rat embryonic hippocampal neurons for different purposes. Although NB medium leads to enhanced cell survival, it contains biological antioxidants and is not suitable for the study of free radical damage and oxidation in cultured neurons. MEM+ without additional antioxidants has been used widely in the study of free radical damage and oxidation, although it does not support optimum neuronal survival in culture. Serum in MEM+ leads to enhanced cell survival but also promotes glial cell proliferation. In this study, we used a new combination medium (NM-2) that consists of both NB and MEM+ for growing primary hippocampal and cortical neuronal cultures. NM-2 enhanced neuronal survival 78.9% for dissociated neurons at a density of 50 cells/mm(2) and 83.1% for 100 cells/mm(2), while decreasing glial cell proliferation to 2-3% and completely inhibiting oligodendrocytes. The NM-2 minimized the effectiveness of antioxidants in the medium to the neurotoxin 4-hydroxynonenal. It also decreased neuronal clumping and provided a more even distribution of neurons. Neurons survived for 4 weeks in NM-2 without changing the original medium. NM-2 provides a good environment for studies of free radical damage and oxidation of neurons. The combination incorporates the best of both NB and MEM+ that results in high neuron survival rate, low glial cell proliferation, reduced antioxidant level, and provides relatively pure cultures of hippocampal and cortical neurons.     

Influence of culture medium and protein supplementation on in vitro oocyte maturation and fertilization in the domestic cat

Domestic cat oocytes were cultured either in Waymouth MB 753/1 Medium (WAY) or in Eagle's Minimum Essential Medium (MEM) containing FSH, LH and estradiol-17beta and supplememted with one of the following: 5% fetal calf serum (FCS); 4 mg/ml bovine serum albumin (BSA); or 3 mg/ml polyvinylalcohol (PVA, a non-protein control). The oocytes were evaluated for: nuclear maturation after 48 hours of culture (in vitro maturation, IVM); fertilization and cleavage 24 to 30 hours postinsemination (in vitro fertilization, IVF); and early embryo development 48 hours postinsemination. Maturation rates were similar (P>0.05) for WAY + BSA (29.4%), MEM + BSA (46.7%) and MEM + PVA (43.3%), but were different (P<0.05) from the other treatments (range, WAY + FCS, 9.6% to WAY + PVA, 14.9%). Fertilization and cleavage rates were also similar (P>0.05) for WAY + BSA (51.4%, 30.5%), MEM + BSA (45.8%, 40.1%) and MEM + PVA (56.1%, 37.4%) and were greater (P<0.05) than all other treatments. These IVM/IVF oocytes were capable of culturing beyond 2-cells, with the highest proportion of 4- and 8- cell embryos forming in WAY and MEM media in the presence of BSA or in MEM medium containing PVA. In the domestic cat IVM/IVF system: both the type of culture medium and protein supplement influence the proportion of oocytes reaching Metaphase II; the type of protein supplement has a more significant (P<0.05) impact than medium on fertilization, cleavage and early embryo development; and nuclear maturation and fertilization in vitro can proceed in this species in the absence of supplementary protein.     

Biological interactions between cell membranes and glycerol or DMSO.

Human erythrocytes washed with phosphate buffered saline (PBS) were frozen for 1 or 16 min at temperatures ranging from ?10 to ?80 °C. Red cell suspensions contained either no protective agent or various concentrations of dimethylsulfoxide (DMSO) or glycerol. The similarities between cryoprotection by DMSO and glycerol reinforce Rapatz and Luyet's classification of cryoprotective agents into three types and support Mazur's two-factor theory of cryoprotection. However, there are important differences between the cryoprotective effects of DMSO and glycerol. The most noteworthy is that for all concentrations of DMSO a 16-min freezing exposure was equal to or more damaging than a 1-min exposure; the converse was true for 11.8 and 17.7% glycerol solutions. This and other differences suggest that the general mechanism of freeze-thaw damage and cryoprotection is more complex than described by Mazur's two-factor theory. Likewise cryoprotective agents cannot be consistently classified into two or three types. A multifactor theory was suggested as a more extensive model for understanding freeze-thaw damage and cryoprotection. The major new contribution of this theory is the idea of biological interaction. This latter refers to solutes in conjunction with various factors which disturb the steady state of the cell membrane. The change in the membrane may be reversible or irreversible depending upon the circumstances.     

Immunotherapy of EL4 lymphoma with reovirus

Summary EL4 lymphoma was grown as an ascitic tumor in the peritoneal cavity of C57Bl/6 mice. Animals with different tumor burdens (either 10⁷ or 10⁹ cells) were treated with a single intraperitoneal injection of BCNU using doses from 20–40 mg/kg. Response as measured by mean survival time and percent survival was dependent on tumor burden and dose of drug. The objective of chemotherapy was to increase the mean survival time, but not the percent survival, in order to evaluate the therapeutic effect of reovirus. Mice were given 10⁸, 10⁹, or 10¹⁰ Pfu of reovirus at various times with respect to chemotherapy. The number of mice cured after treatment with both BCNU and reovirus was significantly greater compared to mice treated with BCNU only. Mice cured with combination therapy developed tumor-specific immunity as measured by cytotoxic lymphocytes and serum, and resistance to a lethal tumor challenge. The Abbreviations used are: BCNU: 1,3-bis-(2-chloroethyl)-1-nitrosourea; Saline: 0.9% NaCl solution; MEM: minimal essential medium; Pfu: plaque-forming units; FCS: fetal calf serum; BME: basal eagle's medium; SSC: sodium citrate-sodium chloride     

Beneficial effect of low concentrations of cryoprotective agents on short-term rabbit kidney perfusion

This report describes observations concerning the influence of the addition of 0.3 M cryoprotective agents (propylene glycol or glycerol) to Ringer's albumin solution used for rabbit kidney perfusion for periods of less than 4 and 24 hr. Endogenous creatinine clearance during short-term parabiotic perfusion on a shunt was used to evaluate function after perfusion. The findings were (A) Perfusion for less than 4 hr resulted in a significant loss of function by comparison with 1 hr ice-stored controls. (B) Continuing the perfusion for 24 hr resulted in a further significant fall in function. Much of the early perfusional injury seen in these two groups could be avoided by including 0.3 M cryoprotective agents in the perfusate.     

Cryopreservation of keratinocytes in a monolayer.

The cryopreservation of cells in tissues is one of the major challenges in current cryobiology, especially with regard to the progressively increasing field of tissue engineering. It is very questionable whether protocols which were developed for the cryopreservation of isolated cells are also applicable for cells in more complex structures, such as tissues. As a starting point toward cryopreservation of these three-dimensional structures, the aim of this study was to find an optimum cryopreservation protocol for keratinocytes in a monolayer (two-dimensional structure). These epidermal cells can be transplanted as a monolayer grown on an appropriate matrix for the treatment of deep-dermal burns and leg ulcers. The successful cryopreservation of such transplants would offer the advantage of long-term storage and immediate availability of the transplant. In our study, the variables investigated were the cryoprotective solution and the cooling rate. In order to find a nontoxic cryoprotective agent (CPA) which could be transplanted without an additional washing step, we included hydroxyethyl starch (HES) as a possible CPA in our experimental protocol with the commonly used CPAs Me(2)SO, glycerol, and ethylene glycol. For the evaluation, the cell survival rate was determined by dye exclusion (trypan blue) and the cell metabolism was investigated by cell activity assay (alamarBlue). In conclusion, the cryopreservation protocol with 10 wt.-% HES resulted not only in the highest survival rate (72%) but also in the highest metabolic activity of the cells after thawing; comparable values for the other CPAs were: Me(2)SO, 48%; glycerol, 8%; and ethylene glycol, 10%.     

Comparison of actual vs. synthesized ternary phase diagrams for solutes of cryobiological interest

Phase diagrams are of great utility in cryobiology, especially, those consisting of a cryoprotective agent (CPA) dissolved in a physiological salt solution. These ternary phase diagrams consist of plots of the freezing points of increasing concentrations of solutions of cryoprotective agents (CPA) plus NaCl. Because they are time-consuming to generate, ternary diagrams are only available for a small number of CPAs. We wanted to determine whether accurate ternary phase diagrams could be synthesized by adding together the freezing point depressions of binary solutions of CPA/water and NaCl/water which match the corresponding solute molality concentrations in the ternary solution. We begin with a low concentration of a solution of CPA+salt of given R (CPA/salt) weight ratio. Ice formation in that solution is mimicked by withdrawing water from it which increases the concentrations of both the CPA and the NaCl. We compute the individual solute concentrations, determine their freezing points from published binary phase diagrams, and sum the freezing points. These yield the synthesized ternary phase diagram for a solution of given R. They were compared with published experimental ternary phase diagrams for glycerol, dimethyl sulfoxide (DMSO), sucrose, and ethylene glycol (EG) plus NaCl in water. For the first three, the synthesized and experimental phase diagrams agreed closely, with some divergence occurring as wt% concentrations exceeded 30% for DMSO and 55% for glycerol, and sucrose. However, in the case of EG there were substantial differences over nearly the entire range of concentrations which we attribute to systematic errors in the experimental EG data. New experimental EG work will be required to resolve this issue.     

Production of buffalo embryos using oocytes from in vitro grown preantral follicles

The present study examines the use of buffalo preantral follicles as a source of oocytes for in vitro embryo production. Preantral follicles were isolated from abattoir-derived buffalo ovaries and were grown for 100 days in five different culture systems: (1) minimum essential medium (MEM); (2) coconut water; (3) MEM + ovarian mesenchymal cell (OMC) co-culture; (4) MEM + granulosa cell (GC) co-culture; or (5) MEM + cumulus cell (CC) co-culture. Low growth rates for the preantral follicles were observed when follicles were cultured in MEM or coconut water medium. Moderate growth rates were seen for OMC and GC co-cultures, and high rates of growth were observed when follicles were grown in CC co-culture. The survival of preantral follicles was low in the MEM culture (<25%), but was over 75% in the other culture systems. Oocytes were not recovered from the MEM group, while an oocyte recovery rate of 80-100% was observed when the follicles were cultured with coconut water/somatic cells. Transferable embryos could be produced only with the oocytes obtained from preantral follicles grown in the OMC and CC co-culture systems. This study demonstrates, for the first time, that it is possible to produce buffalo embryos by in vitro fertilization of oocytes derived from in vitro grown preantral follicles.     

Relationship between proliferative activity and cellular hormono-dependence in the MCF-7 breast cancer cell line

Research on kinetic and hormonal features of breast cancer has led to the development of indices which either reflect accurately the prognosis (incorporation of tritium labelled thymidine) or predict the response to hormonal treatment (presence and concentration of estrogen and progesterone receptors). However, the relationship between cellular proliferation and tumour hormono-dependence has been little studied so far. We describe this relationship in the hormone-dependent MCF-7 cell line cultured in monolayers in MEM + 10% FCS or MEM + 10% FCS (s). We have found that: 1) cellular proliferation and estrogen or progesterone receptor concentration were mutually dependent, the greatest estradiol binding capacity was obtained in cells in which mitotic activity had been slowed down (G0/G1) by the antiestrogenic action of hydroxytamoxifen added to the culture; 2) the presence of estradiol in the culture medium induced marked changes in the synthesis and catabolism of estrogen and progesterone receptors; and 3) both receptors acted as functional proteins whose intracellular concentrations varied depending on the phases of the mitotic cycle.     

A METHOD FOR THE CRYOCONSERVATION OF DUNALIELLA SALINA (CHLOROPHYCEAE): EFFECT OF GLYCEROL AND COLD ADAPTATION1

The unicellular green alga Dunaliella salina Teod. was frozen according to the following procedure: 3 days cold adaptation at 4°C, addition of 3.5 M glycerol as a cryoprotectant, slow cooling to –40°C, immersion in liquid nitrogen, and rapid thawing. The survival rate was higher when cells were grown, before freezing, in the presence of 2 M NaCl instead of 1 M NaCl (78 and 48% survival, respectively). This difference is probably due to the intracellular amount of glycerol, which increases with external NaCl concentration and, therefore, may enhance cell protection. Although cells grown in 4 M NaCl accumulated a large amount of glycerol in response to osmotic stress, they did not withstand freezing. The use of cryoprotectant was absolutely necessary for the cells to recover from storage at –196°C. Glycerol was used because it is naturally produced by Dunaliella salina and therefore is not toxic. Provided it was added slowly to avoid osmotic shock, 3.5 M glycerol gave better results than 1M glycerol (48 and 18% survival, respectively). Cold adaptation in the dark increased postthaw viability. Cells grown in 1 M or 2 M NaCl had a survival rate of 48 and 78%, respectively, when cold-adapted, against 10 and 42% when not cold-adapted. This adaptation could be due to the synthesis, at low temperature, of specific proteins because two bands (28–29 kDa) appeared when electrophoretically separated proteins from cold-adapted cells and control cells were compared. Also, it could be due to the degradation of starch that occurs in the dark and leads to glycerol accumulation. Our procedure has never been used to cryopreserve microalgae and could enhance reported survival rates.     

Effect of cryoprotectants on certain seminal attributes and on the fertility of buck spermatozoa

Semen samples were obtained from 12 bucks (3 Beetal, 3 Black Bengal and 6 Beetal x Black Bengal) and 10 different extenders were constituted with varying concentrations of glycerol, DMSO, glycerol + DMSO and glycerol + lactose as the sperm cryoprotective agents. After the collection of semen samples, they were assessed for quality, diluted in different extenders after removal of seminal plasma, packaged in ministraws and frozen after equilibration (5'C) for 5 h. The samples were evaluated immediately after equilibration and again 24 h after freezing for progressive motility, percentage of live spermatozoa and acrosome, head and tail abnormalities. Both motility and the percentage of live spermatozoa were most affected by extenders containing only DMSO and these values improved in glycerol + DMSO extenders as the concentration of glycerol was increased while DMSO was decreased. However, these values were significantly higher in extenders containing glycerol + lactose as the cryoprotective agents, and were found to increase with increased concentration of lactose, being highest in TYGL (180). Acrosomal and tail abnormalities tended to increase between post equilibration and post thawing stage, and were higher in extenders containing the higher levels of DMSO. Significantly (P < 0.01) lower percentages of abnormalities were recorded in the glycerol + lactose extenders. The fertility results showed nonsignificant effect of extenders on the conception rate of does.     

The mechanism of the cryoprotective effect of glycerol in beetles tolerant to freezing

The mechanism of the cryoprotective effect of glycerol has been studied experimentally on freezetolerant Pytho depressus beetles. The lowest tolerated temperature of the beetles was determined as a function of their pre-freezing glycerol concentration. The temperatures, at which the potentially harmful non-penetrating solutes would reach the injurious level in the frozen body fluid were estimated for solutions with different pre-freezing concentrations of glycerol, assuming a colligative effect of glycerol. The lowest tolerated temperature of the beetles ranged from ?7.5 C for beetles lacking glycerol to ?27 C for beetles having a pre-freezing glycerol concentration of 1500 mmolal in their body fluid. The estimated temperatures fitted these observations almost perfectly. Thus, the results support the view that the cryoprotective effect of glycerol in freeze-tolerant beetles is based on the colligative properties of the substance.     

Differential Expressions of Two Types of Alkaline Phosphatase during Developmental Growth of Embryoid Bodies of Mouse Teratocarcinoma OTT-6050 In Vitro

Ascites teratocarcinoma OTT-6050 is a totipotent tumor line producing indefinitely the simple type of embry-oid bodies (EBs). In culture with fetal calf serum (FCS) in Eagle's minimal essential medium (MEM), these EBs show developmental growth, only in which some differentiative events result. EBs also show this developmental growth in MEM supplemented with two fractions of FCS separated with a Amicon PM 10 membrane, i.e. a low molecular weight Fraction L (mol. wt. less than 10,000) and a high molecular weight Fraction H (mol. wt. more than 10,000). Fraction H is necessary for the survival of EBs in vitro. Fraction L enhances the uptake of ³H-thymidine into EB cells with increase in the Vmax , but no change in the K m. On culture of EBs with both Fractions, a marked bimodal increase in alkaline phosphatase (ALPase) activity is seen on day 1–2 and 4, resulting from the differential expressions of two electrophoretically distinct ALPases (Bands I and II). The differential expressions of ALPase are also observed cytochemically, one activity being on the inner cells and the other on the surrounding cells of EBs. From the cytochemical similarity of ALPase activity to that of normal mouse embryos, Band I ALPase is inferred to be the epiblast (developmentally totipotent stem cell)-type and Band II ALPase to be the distal (parietal) endoderm-type.     

The Effect of Freezing Rate on the Survival of Cryopreserved African Sharptooth Catfish (Clarias gariepinus) Spermatozoa

Cryoprotective agents were evaluated to find the optimal concentration of the cryoprotectant and most suitable combination of solution and cryoprotectant. A cryoprotective agent composed of 4% glucose and 9% glycerol yielded the best results. It was established that the optimal freezing rate is dependent on the composition of the cryoprotective agent. Maximal survival of catfish spermatozoa (60%) occurs at 5°C min^-1 and faster and slower freezing rates result in poor survival or no survival at all. Incorporation of an isothermal holding period into the freezing rate led to remarkable increase (20-30%) in sperm survival when Me₂SO was present in the cryoprotective agent. Cryoprotective agents containing glucose also showed improved survival when a three phase freezing rate was used. These results lead to the conclusion that the presence of an isothermal holding period in the freezing rate is beneficial for the cryoprotective action of Me₂SO and glucose.     

Alterations in pH and Serum Concentration have Contrasting Effects on Normal and "Foreign" Pathways of Differentiation in Cultures of Embryonic Chick Neuroretinal Cells

Nine-day chicken embryo neuroretinal cells transdifferentiate into both lens and pigment cells after 3–4 weeks when cultured in MEM medium containing 10% foetal calf serum at pH 7.4. At pH 6.8. the appearance of lens crystallins is retarded and cholineacetyltransferase (CAT) activity persists for longer, whereas at pH 8.0 crystallins appear earlier and CAT activity declines more rapidly. Cell survival and culture growth are about 10% lower at pH 6.8 than at pH 8.0. If the concentration of foetal calf serum (FCS) is increased from 10% to 25% (at pH 7.4), cell survival and growth are both promoted, crystallins appear slightly earlier and CAT activity declines more rapidly. Converse effects are observed with 5 % serum, accumulation of crystallins being greatly inhibited and CAT activity prolonged. Crystallin production in cultures with 10% or 25% chicken serum (CS) is much less extensive than in similar FCS cultures, but in cultures with 5 % CS, crystallins appear more rapidly, reaching higher levels than in 5 % FCS cultures. However, the pattern of CAT activity in response to different serum levels is similar for both CS and FCS. This might imply the presence of some factor(s) able to stimulate transdifferentiation in FCS, whereas CS can apparently inhibit this process.