二甲基亚砜作用下重组CHO细胞胞内乙型肝炎表面抗原的定位

在培养环境中添加二甲基亚砜 (DMSO) 能分别提高重组 CHO 细胞乙型肝炎表面抗原（HBsAg）的产量和比生产速率70%和3.2倍以上, 但同时发现胞内HBsAg 的积累量是对照组的7.2倍。为了分析胞内HBsAg 积累的区域,采用电镜技术分析后发现,经DMSO处理后的CHO细胞胞内出现了很多的扩张区域,这些扩张区域分布整个胞浆,有的扩张区域已经侵蚀到细胞核上,而对照组未发现明显的扩张区域。进一步利用免疫电镜技术分析后发现,经DMSO处理后的细胞胞内大量积累的HBsAg主要分布在这些扩张区域中,同时发现在细胞核膜上也有分布,这可能是由于扩张区域侵蚀细胞核造成的。以上工作有助于揭示在DMSO作用下重组CHO细胞胞内HBsAg大量积累的机制。     

外源PDI基因导入CHO细胞及其对HBsAg分泌的影响

在前期的研究工作中发现,当培养基中添加1.5%DMSO(二甲基亚砜)会使CHO细胞的HBsAg(乙肝表面抗原)产量提高80%以上。与此同时,DMSO引起了HBsAg在细胞胞内的大量积累,其含量提高了8倍。同时,DMSO引起了CHO细胞中PDI(二硫键异构酶)含量降低。由此怀疑是由于PDI不足造成了HBsAg的胞内积累。根据NCBI上查阅到的中华仓鼠小肠中PDI基因序列,通过RT-PCR的方法,从CHO细胞中获得了PDI序列,并构建重组质粒pGFP-PDI。通过阳离子脂质体转染技术,导入外源PDI,使各单克隆CHO细胞的PDI酶活提高30%～90%。但分析这些克隆在1.5%DMSO下HBsAg的胞内积累情况,发现没有改善,从而排除了PDI不足而引起外源蛋白积累的可能。     

Reduced secretion of virions and hepatitis B virus (HBV) surface antigen of a naturally occurring HBV variant correlates with the accumulation of the small S envelope protein in the endoplasmic reticulum and Golgi apparatus

We identified two novel naturally occurring mutations (W74L and L77R) in the small S envelope protein of hepatitis B virus (HBV). Mutation L77R alone resulted in >10-fold-reduced secretion of virions. In addition, the 2.8-fold reduction of the extracellular HBV surface antigen (HBsAg) of mutant L77R from transfected Huh7 cells appeared to be correlated with a 1.7-fold reduction of intracellular HBsAg, as measured by enzyme-linked immunosorbent assay (ELISA). Surprisingly, opposite to the ELISA results, Western blot analysis revealed a near-10-fold-increased level of the intracellular mutant small S envelope protein. The discrepancy between ELISA and Western blot data was due to significant accumulation of the mutant L77R HBsAg in the intracellular pellet fraction. In contrast to HBsAg, the secretion of HBeAg was normal in L77R-transfected cells. The wild-type HBsAg was usually more diffuse and evenly distributed in the cytoplasm, often outside the perinuclear endoplasmic reticulum (ER) and Golgi apparatus, as observed by immunofluorescence assay. In contrast, the L77R mutant HBsAg tends to be highly restricted within the ER and Golgi, often accumulated in the Golgi compartments distal from the nucleus. The almost exclusive retention in the ER-Golgi of L77R HBsAg was similar to what was observed when the large envelope protein was overexpressed. These multiple aberrant phenotypes of mutant L77R can be corrected by a second naturally occurring S envelope mutation, W74L. Despite the accumulation of L77R HBsAg in ER-Golgi of transfected Huh7 cells, we detected no increase in Grp78 mRNA and proteins, which are common markers for ER stress response.     

Intracellular assembly and packaging of hepatitis B surface antigen particles occur in the endoplasmic reticulum.

Hepatitis B surface antigen (HBsAg) particles are secreted by Chinese hamster ovary cells that are stably transfected with the S gene of hepatitis B virus. The assembly of HBsAg into cylindrical and spherical particles occurred intracellularly within the endoplasmic reticulum. HBsAg particles accumulated within large dilated areas of the endoplasmic reticulum and remained within these structures for most of the time prior to secretion from the cells. Once the particles were formed, the HBsAg polypeptides did not appear to become associated with subsequent intracellular organelle membranes or the plasma membrane. HBsAg within the dilated structures did not bind wheat germ agglutinin, indicating that its oligosaccharide chains had not yet been processed to the complex form (containing terminal sialic acid-N-acetylglucosamine residues). The oligosaccharide chains of HBsAg are processed to the complex form and can be detected on the HBsAg after secretion, but this event was not detected within cells. In addition, HBsAg was not observed on the cell surface by indirect immunofluorescence or immunoprecipitation, although immunoelectron microscopy revealed some staining at or near the cell surface. These results suggested that HBsAg was either secreted from cells without being incorporated into the plasma membrane, or that the levels of HBsAg in the plasma membrane were below the limits of detection.     

Endocytosis of hepatitis B immune globulin into hepatocytes inhibits the secretion of hepatitis B virus surface antigen and virions

Hepatitis B immunoglobulin is used for prophylaxis against hepatitis B virus (HBV) and is thought to act by neutralization of virions and hepatitis B virus surface antigen (HBsAg)-containing particles in circulation. Using a panel of hepatocyte-derived cell lines, the present study investigated in vitro whether HBs-specific immunoglobulin G (IgG) is internalized in hepatocytes and whether it interacts with HBsAg in the cells. By immunoelectron microscopy and immunoblotting, human IgG and FcRn receptor for IgG were demonstrated on cellular membranes and in cytoplasmic extracts, irrespective of the HBsAg status of the cells. Furthermore, HBsAg and anti-HBs were shown to be colocalized in the same cellular compartment by two-color confocal microscopy. Endocytosis of HBs-specific IgG caused intracellular accumulation of HBsAg in a dose-dependent manner and inhibited the secretion of HBsAg and HBV virions from the cells. These effects were not observed with F(ab)(2) fragments or nonimmune IgG as controls. The specificity of intracellular HBsAg- anti-HBs interaction was further investigated in cells transfected with HBV genomes expressing wild-type HBsAg or immune escape HBsAg (with a G145R mutation). Monoclonal anti-HBs markedly reduced the secretion of wild-type HBsAg, while the secretion of mutant HBsAg was not affected. These results suggest that HBs-specific IgG binds to hepatocytes and interacts with HBsAg within the cells. This may be relevant for the selection of surface antibody escape mutations.     

Enhanced human thrombopoietin production by sodium butyrate addition to serum-free suspension culture of bcl-2-overexpressing CHO cells

When sodium butyrate (NaBu) was added to serum-free suspension culture of recombinant CHO (rCHO) cells for enhanced expression of human thrombopoietin (hTPO), apoptotic cell death of rCHO cells was induced in a dose-dependent manner and hTPO quality was deteriorated in regard to sialic acid and acidic isoform contents. To overcome these problems, we overexpressed Bcl-2 protein, an antiapoptotic protein, in rCHO cells producing hTPO. Compared to serum-free suspension culture of control cells without Bcl-2 overexpression (R-neo cells) and NaBu addition, a more than 10-fold increase in the maximum hTPO concentration was obtained in serum-free suspension culture of cells with Bcl-2 overexpression (R-bc12-14 cells) and 3 mM NaBu addition. Both the enhanced specific productivity endowed by NaBu and the extended culture longevity provided by the antiapoptotic effect of Bcl-2 overexpression contributed to the enhancement of maximum hTPO concentration. The problem of quality reduction of hTPO induced by NaBu was not solved by Bcl-2 overexpression, but it was not that significant. Compared to the culture in the absence of NaBu, the percentage of hTPO isoforms in pI 3-5 with high in vivo biological activity produced by R-bc12-14 cells was decreased by approximately 18% in the presence of 3 mM. As a result, a more than 6-fold increase in the production of hTPO isoforms in pI 3-5 was achieved in R-bcl2-14 cell culture with 3 mM NaBu addition. Taken together, the data obtained suggest that Bcl-2 overexpression in rCHO cells and NaBu addition in serum-free suspension culture can be an effective means to enhance the production of highly glycosylated protein such as hTPO.     

Proteomic understanding of intracellular responses of recombinant Chinese hamster ovary cells cultivated in serum-free medium supplemented with hydrolysates

In order to understand the intracellular responses in recombinant CHO (rCHO) cells producing antibody in serum-free medium (SFM) supplemented with optimized hydrolysates mixtures, yielding the highest specific growth rate (μ, SFM#S1) or the highest specific antibody productivity (q _Ab, SFM#S2), differentially expressed proteins in rCHO cells are measured by two-dimensional gel electrophoresis combined with nano-LC-ESI-Q-TOF tandem MS. The comparative proteomic analysis with basal SFM without hydrolysates revealed that the addition of hydrolysate mixtures significantly altered the profiles of CHO proteome. In SFM#S1, the expression of metabolism-related proteins, cytoskeleton-associated proteins, and proliferation-related proteins was up-regulated. On the other hand, the expression of anti-proliferative proteins and pro-apoptotic protein was down-regulated. In SFM#S2, the expression of various chaperone proteins and proliferation-linked proteins was altered. 2D-Western blot analysis of differentially expressed proteins confirmed the proteomic results. Taken together, identification of differentially expressed proteins in CHO cells by a proteomic approach can provide insights into understanding the effect of hydrolysates on intracellular events and clues to find candidate genes for cell engineering to maximize the protein production in rCHO cells.     

Calnexin overexpression sensitizes recombinant CHO cells to apoptosis induced by sodium butyrate treatment

Sodium butyrate (NaBu) can enhance the expression of foreign genes in recombinant Chinese hamster ovary (rCHO) cells, but it can also inhibit cell growth and induce cellular apoptosis. In this study, the potential role of calnexin (Cnx) expression in rCHO cells treated with 5 mM NaBu was investigated for rCHO cells producing tumor necrosis factor receptor FC. To regulate the Cnx expression level, a tetracycline-inducible system was used. Clones with different Cnx expression levels were selected and investigated. With regard to productivity per cell (q_p), NaBu enhanced the q_p by over twofold. Under NaBu treatment, Cnx overexpression further enhanced the q_p by about 1.7-fold. However, under NaBu stress, the cells overexpressing Cnx showed a poorer viability profile with a consistent difference of over 25% in the viability when compared to the Cnx-repressed condition. This drop in the viability was attributed to increased apoptosis seen in these cells as evidenced by enhanced poly (ADP-ribose) polymerase cleavage and cytochrome C release. Ca²⁺ localization staining and subsequent confocal imaging revealed elevated cytosolic Ca²⁺ ([Ca²⁺]_c) in the Cnx-overexpressing cells when compared to the Cnx-repressed condition, thus endorsing the increased apoptosis observed in these cells. Taken together, Cnx overexpression not only improved the q_p of cells treated with NaBu, but it also sensitized cells to apoptosis.     

HBV infection of cell culture: evidence for multivalent and cooperative attachment

Hepadnaviruses do not infect cultured cells, therefore our knowledge of the mechanism of the early stages of virus-cell interaction is rather poor. In this study, we show that dimethylsulfoxide (DMSO)-treated HepG2 hepatoblastoma cells are infected efficiently by serum-derived hepatitis B virus (HBV) as monitored by viral gene expression and replication markers. To measure virus attachment, a variety of HBV surface proteins (HBsAgs) were conjugated to polystyrene beads and their capacity to attach cells was visualized and quantified by light microscopy at a single-cell resolution. Remarkably, DMSO increases the attachment efficiency by >200-fold. We further identify the QLDPAF sequence within preS1 as the receptor-binding viral domain epitope. Interestingly, a similar sequence is shared by several cellular, bacterial and viral proteins involved in cell adhesion, attachment and fusion. We also found that the small HBsAg contains a secondary attachment site that recognizes a distinct receptor on the cell membrane. Furthermore, we provide evidence in support of multivalent HBV attachment with synergistic interplay. Our data depict a mechanistic view of virus attachment and ingestion.     

Proteomic understanding of intracellular responses of recombinant chinese hamster ovary cells adapted to grow in serum‐free suspension culture

To understand the intracellular responses in recombinant Chinese hamster ovary (rCHO) cells adapted to grow in serum‐free suspension culture, a proteomic approach was employed. After rCHO cells producing erythropoietin were adapted to grow in suspension culture with the two different serum‐free media (SFM4CHO? and SF‐L1), proteome analyses were carried out using 2‐D PAGE and based on spot intensities, 58 high‐intensity protein spots were selected. Of the 58 protein spots, which represented 34 different kinds of proteins, 55 were identified by MALDI‐TOF‐MS, and MS/MS. Compared with the results in serum‐containing medium, six proteins, four de novo synthesis of nucleotides‐related proteins (dihydrolipoamide S‐acetyltransferase, transaldolase, inosine‐5′‐monophosphate dehydrogenase 2, and lymphoid‐restricted membrane protein) and two molecular chaperones (heat shock protein 70 kDa and 60 kDa [HSC70, HSP60]) were significantly increased in SFM4CHO?. From the results of proteomic analysis, HSP60 and HSC70, which were increased in both SFM, were selected as candidate proteins for engineering and rCHO cell lines overexpressing these genes were constructed. Cells overexpressing HSP60 and/or HSC70 showed 10–15% enhanced cell concentration during serum‐free adaptation and 15–33% reduction in adaptation time. Taken together, identification of differentially expressed proteins in rCHO cells by a proteomic study can provide insights into understanding the intracellular events and clues to find candidate genes for cell engineering for improved performance of rCHO cells during adaptation to serum‐free suspension culture. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Insulin increases the cell surface concentration of alpha 2-macroglobulin receptors in 3T3-L1 adipocytes. Altered transit of the receptor among intracellular endocytic compartments

The present study shows that insulin causes an increase in the binding of alpha 2-macroglobulin (alpha 2M) to 3T3-L1 adipocytes. Scatchard analysis of the binding at 4 degrees C indicated an approximate 2-fold increase in the number of alpha 2M binding sites, with no change in the apparent affinity of the receptor. In addition, a 2-3-fold increase in the binding of monoclonal antibody 2C6, which recognizes a component of the alpha 2M receptor, was found in cells treated at 37 degrees C with insulin and then KCN to inhibit receptor endocytosis. An increased cellular accumulation of alpha 2M was also observed in response to insulin. Interestingly, the increase in the rate of accumulation of alpha 2M was significantly smaller than the increase in the number of alpha 2M receptors on the cell surface, suggesting that the rate of ligand internalization or subsequent processing is altered in response to insulin. Ultrastructural analysis of the internalization pathway of the alpha 2M receptor was performed using colloidal gold-coupled 2C6 monoclonal antibody. Control cells incubated for 20 min at 37 degrees C with the gold-conjugated antibody displayed 40% of cellular gold particles on the cell surface and 60% within intracellular structures. In insulin-treated cells this proportion was reversed, with 64% of the particles being found on the cell surface, and only 36% within intracellular structures. Significant differences in the distribution of gold particles among intracellular structures were detected between control and insulin-treated cells. Whereas in control cells, 18% of the total cellular gold particles internalized into tubulovesicles and multivesicular bodies, in insulin-treated cells only 3% of the gold particles were found within these structures. These data indicate that the movement of this receptor between endocytic compartments is altered in response to insulin, and suggest that the effect of insulin to increase the cell surface concentration of alpha 2M receptors and the accumulation of alpha 2M is due, at least in part, to alterations in the endocytic portion of the receptor recycling pathway.     

Effect of dimethyl sulfoxide on mouse embryo fibroblasts: inhibition of plasminogen activator inhibitor deposition and interference with early events of serum-stimulated growth

Quiescent and serum-stimulated cultures of Swiss mouse embryo fibroblasts (MEF) showed alterations in cell morphology including an enlargement in size upon treatment with 2% dimethyl sulfoxide (DMSO). Treatment of MEF and monkey kidney epithelial cells (MK2) with 2% DMSO at the early periods of serum-stimulated growth inhibited RNA, protein and DNA synthesis. DMSO treatment of cells at late stages of serum-stimulated growth (MEF after 1 hr and MK2 cells after 3 hr of stimulation) had little effect on DNA and protein synthesis although cell enlargement occurred in these cells. When the [35S]methionine labelled proteins of the control and the DMSO treated cells were analysed by high resolution polyacrylamide gel electrophoresis, no apparent difference was observed in the pattern of intracellular proteins of these cells. In contrast, the extracellular levels of two serum-induced secreted proteins of MEF (Mr 48,000 and 26,000) were dramatically reduced by DMSO treatment. The DMSO sensitive 48 kDa protein was found to be the major component of the extracellular matrix, while the 26 kDa protein was not. The 48 kDa protein was identified as plasminogen activator inhibitor (PAI-1). Densitometric quantitation showed a gradual accumulation of this protein in the matrix of serum-stimulated cells. The deposition of this protein in the matrix was inhibited by DMSO. Flow-cytometric quantitation of indirect immunofluorescence indicated higher intracellular levels of the 48 kDa protein in fetal calf serum (FCS) + DMSO treated cells, suggesting that the low level of this protein in the medium of DMSO treated cells is probably due to lack of transport of this protein from the cells into the medium.     

Influence of down-regulation of caspase-3 by siRNAs on sodium-butyrate-induced apoptotic cell death of Chinese hamster ovary cells producing thrombopoietin

Sodium butyrate (NaBu) can enhance the expression of foreign protein of recombinant Chinese hamster ovary (rCHO) cells, but it can also inhibit cell growth and induce cellular apoptosis. Thus, the beneficial effect of using a higher concentration of NaBu on foreign protein expression in rCHO cells is compromised by its growth inhibitory and cytotoxic effects. To overcome this cytotoxic effect of NaBu, an expression vector of small interfering RNAs (siRNAs) targeting against caspase-3, a key effector component in apoptosis, was constructed and transfected into rCHO cells producing human thrombopoietin (hTPO). Using this siRNA strategy, rCHO cells (F21 cells) expressing a low level of caspase-3 proenzyme determined by RT-PCR and Western blot analysis were established. Under the condition of 1-5 mM NaBu addition at the exponential growth phase, down-regulation of caspase-3 in F21 cells could not effectively inhibit NaBu-induced apoptotic cell death. This NaBu-induced apoptotic cell death occurred because F21 cells appeared to compensate for the lack of caspase-3 by increasing the active caspase-7 level. These results suggest that the intracellular caspase's interconnectivity should be taken into consideration for the successful inhibition of apoptosis of rCHO cells.     

Effect of doxycycline-regulated calnexin and calreticulin expression on specific thrombopoietin productivity of recombinant Chinese hamster ovary cells

In an attempt to increase the specific thrombopoietin (TPO) productivity (q(TPO)) of recombinant Chinese hamster ovary (rCHO) cells (CHO-TPO), the effect of expression level of calnexin (CNX) and calreticulin (CRT) on q(TPO) was investigated. To control both CNX and CRT expression levels simultaneously, the Tet-Off system was first introduced in CHO-TPO cells, and stable Tet-Off cells (TPO-Tet-Off) were screened by luciferase assay. The doxycycline-regulated CNX and CRT expression system in rCHO cells (TPO-CNX/CRT) was established by cotransfection of CNX and CRT expression vector and pTK-Hyg vector into TPO-Tet-Off cells and subsequent screening by Western blot analysis of CNX and CRT. The expression levels of CNX and CRT in TPO-CNX/CRT cells could be tightly controlled by adding different concentrations of doxycycline to a culture medium. Compared with the basal level (2 microg/mL doxycyline), a 2.9-fold increase in CNX expression and a 2.8-fold increase in CRT expression were obtained in the absence of doxycycline. This, in turn, resulted in a 1.9-fold increase in q(TPO), not inhibiting cell growth or changing in vivo biological activity of TPO. Taken together, these results demonstrate that a simultaneous overexpression of CNX and CRT can increase the q(TPO) of rCHO cells.     

A role of GADD153 in ER stress-induced apoptosis in recombinant Chinese hamster ovary cells

The imbalance between the folding capacity and the folding demand imposed on the endoplasmic reticulum (ER) of therapeutic protein-producing host cells results in a stressed ER. This initiates a series of cellular signaling events termed the unfolded protein response (UPR) aimed at restoring homeostasis. In order to alleviate ER stress and ER stress-induced apoptosis in recombinant Chinese hamster ovary (rCHO) cells, silencing of the growth arrest and DNA damage 153 gene (GADD153), the main pro-apoptotic factor of UPR, was attempted. The rCHO cells were cultured under four ER stress inducing conditions, including thapsigargin, brefeldin A, glucose deprivation, glucose and glutamine deprivation. In these conditions, the functions of stably GADD153-silenced clones were investigated. It was found that under exclusive ER stress-inducing conditions of thapsigargin and brefeldin A treatments, the GADD153-silenced clones showed a less incidence of apoptosis (about 38%) and less cell viability (about 58% non-viable cells) than the control cells. However, under nutrient deprivation, the beneficial effect of GADD153 silencing was not pronounced because nutrient deprivation led to a cascade of various events including GADD153-induced cell death. GADD153-overexpressing pool cells also substantiated the findings of GADD153 downregulation. Investigation of the underlying mechanism revealed that increased GADD153 expression results in an exaggerated production of reactive oxygen species (ROS) and that GADD153 silencing promotes translational attenuation facilitating cell recovery from stress. Taken together, this study suggests that GADD153 sensitizes cells to ER stress through mechanisms that involve enhanced oxidative injury and by manipulating the ER client protein load in rCHO cells.     

Development of serum-free medium supplemented with hydrolysates for the production of therapeutic antibodies in CHO cell cultures using design of experiments

An efficient method of formulating serum-free medium (SFM) for production of therapeutic antibodies by recombinant CHO (rCHO) cells was developed using two rCHO cell lines producing a therapeutic antibody. In this method, ten kinds of SFM were prepared by supplementing the basal SFM with statistically designed mixtures (total 5 g L^?1) of three non-animal-derived hydrolysates: yeastolate, soy hydrolysate, and wheat gluten hydrolysate. When the two rCHO cell lines were cultivated, the mixtures of soy hydrolysate and wheat gluten hydrolysate showed a positive effect on cell growth. On the other hand, the mixtures including a high portion of yeastolate significantly enhanced specific antibody productivity. To reconstitute the mixture ratios of the three hydrolysates for high growth and antibody production, the effect of each medium was analyzed by the statistical program Design-Expert®. The resulting medium gave a 1.9–3.3-fold increase in the maximum antibody concentration, compared to the basal SFM. Taken together, the supplementation of hydrolysates to the basal SFM with the help of statistical analysis is an efficient means of developing SFM for therapeutic antibody production by rCHO cells.     

Effect of Bcl‐xL overexpression on apoptosis and autophagy in recombinant Chinese hamster ovary cells under nutrient‐deprived condition

Upon nutrient deprivation during culture, recombinant Chinese hamster ovary (rCHO) cells are subjected to two types of programmed cell death (PCD), apoptosis and autophagy. To investigate the effect of Bcl‐x_L overexpression on apoptosis and autophagy in rCHO cells, an erythropoietin (EPO)‐producing rCHO cell line with regulated Bcl‐x_L overexpression (EPO‐off‐Bcl‐x_L) was established using the Tet‐off system. The expression level of Bcl‐x_L in EPO‐off‐Bcl‐x_L cells was tightly regulated by doxycycline in a dose‐dependent manner. Bcl‐x_L overexpression enhanced cell viability and extended culture longevity in batch culture. Upon nutrient depletion in the later stage of batch culture, Bcl‐x_L overexpression suppressed apoptosis by inhibiting the activation of caspase‐3 and ‐7. Simultaneously, Bcl‐x_L overexpression also delayed autophagy, characterized by LC3‐II accumulation. Immunoprecipitation analysis with a Flag‐tagged Bcl‐x_L revealed that Bcl‐x_L interacts with Bax and Bak, essential mediators of caspase‐dependent apoptosis, as well as with Beclin‐1, an essential mediator of autophagy, and may inhibit their pro‐cell death function. Taken together, it was found that Bcl‐x_L overexpression inhibits both apoptosis and autophagy in rCHO cell culture. Biotechnol. Bioeng. 2009;103: 757–766. © 2009 Wiley Periodicals, Inc.     

Freezing of rat lymphocytes. I. The effects of dimethyl sulfoxide and freezing on the phytohemagglutinin- and pokeweed mitogen-responding lymphocyte subpopulations.

Rat spleen and lymph node lymphocytes have been frozen with dimethyl sulfoxide (DMSO) at 1 °C/min and stored at ?196 °C for 10 min. The functional recovery of the cell populations was monitored by the mitogenic response (uptake of [³H]thymidine) to phytohemagglutinin (PHA) or pokeweed mitogen (PWM) in culture after thawing. With 5 to 10% DMSO in the freezing medium, frozen-thawed lymph node cells were found to retain about 40% of their response to PHA. In contrast, frozen-thawed spleen cells responded better to PHA than fresh cells. The response to PWM was markedly decreased in both spleen and lymph node cell cultures.A similar effect was observed when DMSO was added to the culture medium of fresh spleen cells, i.e., an augmentation of the response to PHA and a suppression of the response to PWM. However, the concentrations of DMSO needed to induce this effect was more than 10-fold higher than that present in the culture medium after freezing and thawing.Since removal of adherent cells from the spleen cell population also produced an augmentation of the response to PHA, it is suggested that the freezing procedure and DMSO may have an inhibitory effect on suppressor cell functions present in spleen cell populations.