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.     

Interaction of liposomes with Kupffer cells in vitro

We investigated the interaction of liposomes with rat Kupffer cells in monolayer maintenance culture. The liposomes (large unilamellar vesicles, LUV) were composed of 14C-labelled phosphatidylcholine, cholesterol and phosphatidylserine (molar ratio 4:5:1) and contained either 3H-labelled inulin or 125I-labelled bovine serum albumin as a non-degradable or a degradable aqueous space marker, respectively. After 2-3 days in culture the cells exhibited optimal uptake capacity. The uptake process showed saturation kinetics, maximal uptake values amounting to 2 nmol of total liposomal lipid/h/10(6) cells. This is equivalent to 1500 vesicles per cell. The presence of fetal calf serum (FCS) during incubation increased uptake nearly two-fold, whereas freshly isolated rat serum had no effect. The binding of the liposomes to the cells caused partial release of liposomal contents (about 15-20%) both at 4 degrees C and at 37 degrees C. In the presence of metabolic inhibitors the uptake at 37 degrees C was reduced to about 20% of the control values. Inulin and lipid label became cell-associated at similar rates and extents, whereas the association of albumin label gradually decreased after attaining a maximum at relatively low values. When, after 1 h incubation, the liposomes were removed continued incubation for another 2 h in absence of liposomes led to an approx. 30% release of cell-associated lipid label into the medium in water-soluble form. Under identical conditions as much as 90% of the cell-associated albumin label was released in acid-soluble form. Contrarily, the inulin label remained firmly cell-associated under these conditions. From these results we conclude that Kupffer cells in monolayer culture take up liposomes primarily by way of an adsorptive endocytic mechanism. This conclusion was confirmed by morphological observations on cells incubated with liposomes containing fluorescein isothiocyanate (FITC) dextran or horseradish peroxidase as markers for fluorescence microscopy and electron microscopy, respectively.     

Disappearance of a 32 000 D chromatin polypeptide during early erythroid differentiation of Friend leukemia cells

The patterns of non-histone chromatin proteins have been investigated in dimethyl sulphoxide (DMSO)-stimulated Friend leukemia cells (FLC) undergoing the “early” events of erythroid differentiation. Sucrose-purified whole nuclei were lysed and proteins extracted with 6 M urea and 4 M guanidium hydrochloride. The proteins were analysed in SDS-and in SDS-urea-polyacrylamide gel electrophoresis. The disappearance of a 32 000 D chromatin protein component was observed in cells of the 745A “inducible” line harvested as early as 24 h after DMSO treatment, as compared with untreated 745A cells and to cells harvested 6 and 12 h after DMSO addition to the cultures. By contrast, a 32 000 D chromatin protein component is always present in untreated as well as in DMSO-treated cells of the “uninducible” Fw line of FLC.     

Dimethyl sulfoxide enhances lipid synthesis and secretion by long-term cultures of adult rat hepatocytes.

Dimethyl sulfoxide (DMSO) was tested for its effects on lipid metabolism of long-term cultures of adult rat hepatocytes. The addition of 1% DMSO to 3T3-hepatocyte cultures was not toxic to cells and in fact treated cultures maintained better their characteristic morphology for up to 14 days of exposure. DMSO treatment increased 2-3 fold the de novo synthesis of total lipids from[14C]acetate. The analysis by thin layer chromatography of cellular and secreted lipids revealed that DMSO increased the levels of cellular triglycerides, phospholipides and free and sterified cholesterol at 7 days of exposure while at 14 days there was also a 2-3-fold increase in medium secreted lipids. Additionally, DMSO increased the activity of glycerol-phosphate dehydrogenase, a marker enzyme of glycerolipid synthesis, by greater than 50% at either 7 or 14 days of exposure. These results show that 1% DMSO not only is not detrimental to cultured hepatocytes but also enhances lipid synthesis and secretion, both hepatic-differentiated functions.     

A mouse hepatoma cell line which secretes several serum proteins including albumin and alpha-foetoprotein.

A permanent cell line (BW) was established from a transplantable mouse hepatoma, BW7756, which produces alpha-foetoprotein (AFP). Three clones were isolated from the uncloned culture: BW1, BW2 and BWTG3. The cells of the latter clone, which was isolated after selection in the presence of thioguanine, are deficient in the enzyme hypoxanthine-guanine-phosphoribosyl transferase. Both BW1 and BWTG3 cells have mean chromosome number of 64 (60 telocentric and 4 metacentric chromosomes). All three clones secrete at least five serum proteins into the culture medium: albumin, AFP, and alpha 2 globulin, transferrin and C3, the third component of complement. The approximate rate of albumin secretion by BW1 and BWTG3 cells is 10 mug/24 h/10(6) cells. Both albumin and AFP can easily be detected in cell extracts. The simultaneous production of AFP and a hepatocyte specific marker (albumin) by cloned hepatoma cells show that the production of AFP by the tumour is due to the tumoural hepatocytes themselves.     

A Mouse Hepatoma Cell Line which Secretes Several Serum Proteins Including Albumin and α-foetoprotein

A permanent cell line (BW) was established from a transplantable mouse hepatoma, BW7756, which produces α-foetoprotein (AFP).Three clones were isolated from the uncloned culture: BW1, BW2 and B WTG3. The cells of the latter clone, which was isolated after selection in the presence of thioguanine, are deficient in the enzyme hypoxanthine-guanine-phosphoribosyl transferase. Both B W1 and BWTG3 cells have mean chromosome number of 64 (60 telocentric and 4 metacentric chromosomes). All three clones secrete at least'five serum proteins into the culture medium: albumin, AFP, an a2 globulin, transferrin and C3, the third component of complement. The approximate rate of albumin secretion by BW1 and BWTG3 cells is 10 μg/24 h/10⁶ cells. Both albumin and AFP can easily be detected in cell extracts. The simultaneous production of AFP and a hepatocyte specific marker (albumin) by cloned hepatoma cells show that the production of AFP by the tumour is due to the tumoural hepatocytes themselves.     

The role of bile acids in the reduction in lipopolysaccharide uptake by cultured rat Kupffer cells

The influence of bile salts on the binding and uptake of Salmonella abortus equi lipopolysaccharide by cultured Kupffer cells was studied. In control preparations, the percentage of cell-associated lipopolysaccharide increased with time and reached a plateau after about 2 h incubation at 37 degrees C. About 1.2 micrograms lipopolysaccharide was associated with 10(6) Kupffer cells at this time interval. In the presence of 0.3, 0.6 and 1 mumol bile salts/ml the cell-associated lipopolysaccharide was respectively, about 5%, 13% and 29% lower than in control cultures. In the presence of 1 mumol bile salts/ml, the association of lipopolysaccharide to cells at 0 degrees C was about 25% lower than in controls. Preincubation of Kupffer cells with 1 mumol bile salts/ml, with or without lipopolysaccharide, did not affect cell-associated lipopolysaccharide after removal of the bile salts. The rate of secretion of radioactivity by Kupffer cells was not influenced by the presence of bile salts during the uptake or the secretion periods. Bile acids proved to inactivate lipopolysaccharide. From these observations it was concluded that low concentrations of bile salts influence the binding and uptake of lipopolysaccharide by Kupffer cells. It was, therefore, considered likely that, in patients with obstructive jaundice, the high serum bile acid level accounts for spill-over of portal lipopolysaccharide into the systemic blood.     

Gap junction expression and cell proliferation in differentiating cultures of Cx43 KO mouse hepatocytes

Primary cultures of adult mouse hepatocytes are shown here to reexpress differentiated hepatocyte features following treatment with 2% DMSO and 10(-7) M glucagon. To examine the roles of gap junctional communication during hepatocyte growth and differentiation, we have compared treated and untreated hepatocytes from connexin (Cx)32-deficient [Cx32 knockout (KO)] and wild-type mice. In untreated cultures, DNA replication of Cx32 KO hepatocytes was markedly higher than of wild types. Although Cx26 mRNA levels remained high at all time points in wild-type and Cx32 KO hepatocytes, Cx32 mRNA and protein in wild-type hepatocytes underwent a marked decline, which recovered in 10-day treated cultures. Increased levels of Cx26 protein and junctional conductance were observed in Cx32 KO hepatocytes at 96 h in culture, a time when cell growth rate was high. Treatment with DMSO/glucagon highly reinduced Cx26 expression in Cx32 KO hepatocytes, and such treatment reinduced expression of both Cx32 and Cx26 expression in wild types. Dye transfer was not observed following Lucifer yellow injection into DMSO/glucagon-treated Cx32 KO hepatocytes, whereas the spread was extensive in wild types. Nevertheless, high junctional conductance values were observed in treated cells from both genotypes. These studies provide a method by which the differentiated phenotype can be obtained in cultured mouse hepatocytes and provide in vitro evidence that expression of gap junctions formed of Cx32 are involved in the regulation of growth of mouse hepatocytes.     

Optimization of cryoprotectants for cryopreservation of rat hepatocyte

Rat hepatocytes were cryopreserved in hormonally-defined medium (HDM) containing either fetal bovine serum (FBS), glycerol, dimethyl sulfoxide (DMSO), sucrose or a mixture of these as a cryoprotectant. The best survival was with 10% (v/v) DMSO containing 30% (v/v) FBS using 5 x 10(5) hepatocytes ml(-1) at -70 degrees C for 5 d on type I collagen-coated dishes. After thawing, the cell viability was 81% determined by the MTT-test. The cryopreserved hepatocytes had the capacity of albumin synthesis similar to hepatocytes without cryopreservation. This result shows that cryopreservation of rat hepatocyte can be used for the evaluation of hepatic functions.     

Redifferentiation of proliferated rat hepatocytes cultured in L15 medium supplemented with EGF and DMSO

Summary Primary adult rat hepatocytes were cultured in serum-free L15 medium supplemented with 20 mM NaHCO₃ and 10 ng/ml epidermal growth factor in a 5% CO₂:95% air incubator. The number of cells increased and reached about 180% of the initial value by Day 4, and after 2% dimethyl sulfoxide (DMSO) was added to the culture medium at Day 4, the cells continued to proliferate until Day 6. The number of cells reached about 210% at Day 6 and they were well maintained until Day 18. The cell number gradually decreased with time in culture, but many cells remained for more than 2 mo. On the other hand, without 2% DMSO, the cells proliferated until Day 5, but thereafter they rapidly decreased. After DMSO addition, albumin and transferrin were secreted into the medium and the production of both proteins continued for more than 2 mo. Immunocytochemically both proteins were strongly stained in the cells treated with 2% DMSO. Although the expression of G6Pase in the cells disappeared at Day 6 without DMSO, the cells treated with 2% DMSO recovered G6Pase activity at Day 16. In addition, induction of peroxisomes by 2 mM sodium clofibric acid was clearly shown in the hepatocytes at Day 14 and Day 25 using enzyme-cytochemistry. Ultrastructurally, DMSO-treated hepatocytes had many mitochondria and large peroxisomes with a crystalline nucleoid, and both gap junctions and desmosomes were well developed between the cells even at Day 40. Thus, the number of cells doubled, some differentiated functions of the primary hepatocytes were well restored by the use of 2% DMSO, and these functions were maintained for more than 2 mo.     

Improved production of recombinant tumstatin in stably transformed Trichoplusia ni BTI Tn 5B1-4 cells

We describe the expression and in vitro activity of recombinant tumstatin from stably transformed Trichoplusia ni BTI Tn 5B1-4 cells. Recombinant tumstatin was secreted into a culture medium with a molecular weight of 29 kDa. Recombinant tumstatin was also purified to homogeneity using a simple one-step Ni2+ affinity fractionation. Purified recombinant tumstatin inhibited endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition (ED50) for recombinant tumstatin expressed in stably transformed Tn 5B1-4 cells was approximately 0.76 microg/ml. A maximum production level of 4.0 mg/l recombinant tumstatin was obtained in a T-flask culture of Tn 5B1-4 cells, 6 days after cultivation. We also investigated the individual effects of both dimethyl sulfoxide (DMSO) and sodium butyrate on recombinant tumstatin production in stably transformed Tn 5B1-4 cells. Supplementing cultures with DMSO and sodium butyrate separately increased recombinant tumstatin production in stably transformed Tn 5B1-4 cells by 117 and 32%, respectively.     

Permeabilization and in situ adsorption studies during growth and coumarin production in hairy root cultures of Cichorium intybus L

Effect of addition of a permeabilizing agent dimethyl sulfoxide (DMSO) and a solid adsorbent, XAD -7, on growth and coumarin production in hairy root cultures of C. intybus was studied. Continuous permeabilization of the hairy root cultures of C. intybus with DMSO has been shown to be an effective strategy for enhanced release of coumarins while preserving the root viability. DMSO at 0.2% (v/v) level showed the maximum growth and coumarin production but was less as compared to control on day 28. Treatment of cells with increasing concentrations of DMSO (0.3 - 0.6 % v/v) to hairy root cultures of C. intybus, showed an inverse relationship with growth and coumarin production. Growth and production of coumarins increased with 1% media filtrate (MF) of cultures of Phytopthora parasitica var. nicotiana treatment. It was observed that treatment with DMSO (0.2% v/v) and 1% MF of P. parasitica showed the better growth and coumarin production with an increased release of coumarins as compared to the control and other treatments. It was observed that treatment of hairy root cultures with XAD-7 resulted in lesser growth and coumarin production as compared to control during the culture period. Addition of XAD-7 along with 1% MF of P. parasitica showed enhanced growth, coumarin production and increased adsorption as compared to control and lone XAD-7 treatment. Combined addition of DMSO/XAD-7 with fungal elicitor showed synergistic response in terms of biomass and coumarin production. Excretion of coumarins in both the cases was dependent on the presence of DMSO/XAD-7. These results showed that continuous permeabilization of hairy root cultures of C. intybus by using DMSO at 0.2% (v/v) level coupled with 1% MF of P. parasitica maintained viability of tissues and produced coumarins at higher level.     

Effect of sodium butyrate on S-100 protein levels and the cAMP response

Sodium butyrate (NaB), when added to cell cultures, produces a variety of morphological and biochemical changes. We examined its effects, in nM concentrations, on the expression of two glioma cell-associated proteins, glial fibrillary acidic protein (GFAP) and S-100 protein in human glioma-derived cell line (RF), and of S-100 protein in the C6 rat glioma cell line. GFAP levels decreased by about 50% in the RF cell line, and S-100 protein levels decreased protein levels decreased by about 40% after treatment with 1 mM NaB for 48 h. In the C6 rat glioma cell line, isoproterenol with theophylline was found to increase S-100 levels by two-fold over basal levels. NaB was found to inhibit the induction of S-100 protein but exhibited no effect on the basal levels of the protein. Other short chain fatty acids, including sodium propionate and sodium isobutyrate, exhibited partial inhibitory activity. NaB, at an EC50 of 1 mM, was also found to inhibit both the beta-adrenergic and the forskolin-mediated increase in cAMP levels in these cells. This suggests that NaB may inhibit cells from expressing S-100 protein by attenuating cAMP levels.     

Effect of dimethyl sulfoxide on human carcinoma cells, inhibition of plasminogen activator synthesis, change in cell morphology, and alteration of response to cholera toxin.

Human carcinoma HEp-3 lost its tumorigenic and metastatic potential upon prolonged culture in vitro. This change was accompanied by a reduced production of plasminogen activator (PA) of the urokinase type (uPA), which is secreted by HEp-3 cells, a change in response to effectors that modulate uPA production, and an alteration of cell morphology. Similar but more rapid changes occurred when malignant HEp-3 cells were exposed to dimethyl sulfoxide (DMSO). uPA activity in the culture medium dropped below 50% of the control level within 6 h after the addition of DMSO and became undetectable after 24 h of treatment. This drop in uPA activity was not caused by an increased production of PA inhibitors. The cell-associated uPA decreased to 25 to 30% of the control level within 6 h of DMSO treatment and remained at this level for at least 96 h; the reduced uPA production was partially accounted for by a rapid decrease in the functional and chemical concentration of uPA mRNA. In contrast, the concentrations of most of the abundant mRNA species did not appear to be significantly affected, and cell growth was only slightly inhibited in the presence of DMSO. Malignant HEp-3 cells treated with DMSO responded to cholera toxin with an enhanced production of uPA, and their morphology became indistinguishable from that of nonmalignant HEp-3 cells grown in vitro for prolonged periods of time. All of the above changes were fully and rapidly reversible. The inhibitory effect of DMSO on PA production appears to be specific for uPA of human cell lines.     

Temperature- and dose-dependent internalization of concanavalin A in monolayer culture

When rat hepatoma cells (R117-21B) were incubated for 20 h at 37 degrees C with 125I-labeled concanavalin A at low concentrations (0.5-10 micrograms/ml), only 20-30% of the cell-associated radioactivity was released by alpha-methyl-D-mannoside, but at high concentrations (50-500 micrograms/ml), 60-80% of the cell-associated radioactivity was released. At 4 degrees C, the cell-associated radioactivity decreased with the increase in concentration of concanavalin A, and more than 80% of the cell-associated radioactivity was released by alpha-methyl-D-mannoside. These results suggest that the amount of cell-associated concanavalin A is related to the physicochemical state of the plasma membrane, which can be altered by the incubation temperature or by the concentration of concanavalin A, the transitional concentration being 5-10 micrograms/ml.     

The viability of cryopreserved PBPC depends on the DMSO concentration and the concentration of nucleated cells in the graft

BACKGROUND: DMSO is widely used as a cryoprotectant for PBPC. It is desirable to reduce the amount of DMSO without jeopardizing the quality of the stem cell product. The present study was undertaken to investigate whether recovery and survival of CD34+ cells would be significantly altered when PBPC used for autologous transplantations were cryopreserved with four different DMSO concentrations. METHODS: Apheresis samples of PBPC from 20 consecutive patients were mixed in parallel with 2%, 4%, 5% and 10% DMSO, frozen with identical cell concentrations at a controlled rate, and stored in liquid nitrogen for 6-8 weeks. PBPC samples from 11 consecutive patients were also cryopreserved with two different cell concentrations (150 and 300 x 10(6) nucleated cells/mL) to investigate the effect of increasing the cell concentrations while decreasing the DMSO concentration. The flow cytometric absolute count method, based on ISHAGE guidelines, was used to measure the absolute count of total and viable CD34+ cells in the post-thaw samples. RESULTS: PBPC cryopreserved at 150 x 10(6) cells/mL with 2% DMSO yielded significantly inferior CD34+ cell recovery (P < 0.001) and survival (P < 0.001) compared with cryopreservation with 4% and 5% DMSO. This was also observed when comparing higher cell concentrations. However, a reduced cell survival (P = 0.02) was observed when the nucleated cell concentration was increased from 150 to 300 x 10(6) cells/mL in samples cryopreserved with 5% DMSO. DISCUSSION: We conclude that 5% DMSO may be the optimal dose for cryopreserving PBPC as long as the cells have not been concentrated at much more than 200 x 10(6) nucleated cells/mL.     

Isolation and characterization of chromaffin granules from a pheochromocytoma (PC 12) cell line

Incubation of rat kangaroo PtK2 cells with increasing concentrations of dimethylsulfoxide (DMSO) in the growth medium results in striking rearrangements of actin containing structures. After 1 h at concentrations of DMSO between 7.5 and 15%, immunofluorescence microscopy reveals actin containing inclusions within the nucleus of a large proportion of interphase cells. These paracrystals, which seem identical to those described by Fukui by electron microscopy [1], appear not to contain the microfilament-associated proteins tropomyosin, α-actinin or myosin and disappear within 1 h when the cells are shifted to normal medium. Electron microscopy confirms the intranuclear location. At concentrations above 20% DMSO the cells do not recover upon incubation in DMSO-free medium. When DMSO is present at a concentration of 50% the cells appear fixed, no paracrystals are formed and the actin profile resembles that seen in normal cells. Nuclear actin inclusions which appear similar to those induced by DMSO are also found upon incubation of PtK2 cells with the ionophore A23187 in the presence of high levels of magnesium ions. These conditions also result in striking morphological changes of the PtK2 cells. The data suggest that A23187 and DMSO may affect cellular morphology by changing the permeability of the cell to divalent cations, and that at least some of the actin found in the nuclear inclusions is of cytoplasmic origin.     

Acute-phase-response induction in rat hepatocytes co-cultured with rat liver epithelial cells.

The response of rat hepatocytes co-cultured with rat liver epithelial cells to conditioned medium (CM) from lipopolysaccharide (LPS)-activated monocytes was investigated by measuring the concentration of alpha 2-macroglobulin (alpha 2M), alpha 1-acid glycoprotein (AGP), albumin and transferrin, as well as the changes in glycosylation of alpha 1-acid glycoprotein. During an initial 8-day treatment with CM, concentrations of alpha 2M and AGP increased markedly over those of control culture, whereas concentrations of albumin and transferrin decreased. The glycosylation pattern of AGP indicated an important relative increase of the concanavalin A-strongly-reactive (SR) variant upon treatment. When CM addition to hepatocyte culture medium was stopped, the concentrations of the four proteins and the glycosylation pattern of AGP reverted to those of control cultures. Further addition (on day 15) to cultures of CM increased the concentration of alpha 2M and decreased albumin and transferrin concentrations. Although AGP concentrations did not increase above those of controls, the appearance of the SR variant was again stimulated by CM. These results show that, in co-culture, rat hepatocytes remain able to respond to repeated inflammatory stimuli.     

Differential effects of dimethyl sulfoxide and sodium butyrate on alpha-fetoprotein, albumin, and transferrin production by rat hepatomas in culture

Radioimmunoassay was used to determine alpha-fetoprotein (AFP), albumin, and transferrin production (ng/10(5) cells/24 h) by two cell lines (7777 and 8994) derived from chemically induced rat hepatomas. alpha-Fetoprotein production was high (2000 to 4400) in 7777, but was very low (0.2 to 0.4) in 8994. Albumin production varied from 0.4-0.8 (7777) to 14-26 (8994). Both lines produced substantial amounts of transferrin (180 to 240 by 7777 and 29 to 42 by 8994). Addition of dimethyl sulfoxide (DMSO, 1 to 4%) or sodium butyrate (BA, 0.5 to 2.0 mM) to the medium inhibited growth in both lines, but 8994 was more sensitive to these agents than 7777. Dimethyl sulfoxide treatment (2 to 4%) resulted in a dose-related decrease (less than 10% of control at 4% DMSO) in AFP, albumin, and transferrin production by 7777, but in 8994, DMSO (1 to 2%) resulted in an increase (up to sixfold) in albumin and transferrin production, without affecting AFP production. By contrast, BA (2 to 4 mM) stimulated the production of all three proteins in both lines, most notably that of albumin (up to sixfold) by 7777 and that of AFP (up to 20-fold) by 8994. It is concluded that both DMSO and BA can enhance the expression of differentiated functions of the hepatoma cell, and that DMSO at the same time can suppress the expression of an oncofetal function. However, neither DMSO nor BA is selective in its effects on specific genes (i.e., normal, adult vs. oncofetal genes), and it appears that their effects may be the result of a more general phenomenon, the expression of which may be related to the stage of differentiation of the cell.