Survival and phenol red metabolism in chick embryo hepatocytes cultured in the serum-free medium 199 supplemented with dextran T-500 and antioxidants

The supplementation of serum-free Parker's medium 199 with Dextran T-500 and antioxidants (taurine or catalase) significantly improved spraeding and survival of chick embryo hepatocytes in primary culture. To decrease cell surface injury, the dispersion of liver tissue by colagenase was replaced by dissociation of liver with EGTA solution. In such chemically defined culture conditions hepatocytes were able to glucoronise phenol red better than they did it when cultured in the presence of serum. These results suggest that Dextran T-500 and factors which prevent peroxidation of membrane lipids can simplify the conditions useful for the culture of quiescent hepatocytes in a serum-free media.     

Comparison of three culture media for the establishment of melanoma cell lines

Melanoma cell lines are useful tools for the analysis of tumor-specific lymphocytes which are injected to patients treated by adoptive immunotherapy. So they have been established previously (with an efficacy of 47%) in Roswell Park Memorial Institute (RPMI) medium enriched with fetal calf serum (FCS). In order to improve the probability of establishing melanoma cell lines, we compared two FCS-free media with the original FCS medium. Ten melanoma-invaded lymph nodes were tested for their ability to grow in three different culture media: RPMI with FCS; RPMI with human serum (HS); serum-free X-vivo 15 (X15). For each medium, we compared the following criteria: percentage of lines obtained; period of establishment; cell morphology; expression of melanoma-associated antigens and surface molecules. More cell lines were obtained with HS and X15 media compared to FCS medium (7/10, 5/10 and 4/10, respectively). The time period to establish a stable line was similar for the three media. No morphological differences were observed in cells derived from the same tumor sample in the different media. With the X15 medium, cells generally expressed lower levels of melanocytic differentiation antigens and surface molecules. The growth of melanoma cell lines in FCS-free culture media appears possible and advantageous, with an increased probability of obtaining autologous tumor cell lines. Furthermore the cells obtained could be used as multiple antigenic sources in active or adoptive immunotherapy protocols.     

Establishment of human parotid pleomorphic adenoma cells in culture: Morphological and biochemical characterization

Summary This study reports the establishment ofα-amylase-producing human parotid pleomorphic adenoma cell lines (2HP and 2HP₁) which have been maintained in culture for over 1 yr. The procedures required preparation of cellular clumps from tumor tissue and plating them on plasma clot or precoated dishes. During the initial phase of growth they required modified MCDB-153 medium without serum. When cells showed signs of degeneration they were changed to MCDB-153 medium containing first 2% and then 10% heat inactivated fetal bovine serum. Although cells grew well in MCDB-153 containing 10% serum, the epithelial cell morphology was not distinct. Therefore, the growth and morphology of cells grown in MCDB-10% serum were compared with those in RPMI growth medium containing 10% fetal bovine serum and F12 containing 10% agammaglobulin newborn bovine serum. Although the growth of cells was a little slower in F12 medium than those in MCDB and RPMI, the epithelial cell morphology was maintained better than in other growth media. The cells of 2HP and 2HP₁ produce low levels ofα-amylase and relatively high levels ofα-amylase mRNAs of 1176 and 702 bp and contain neurofilament-160, a neuronal-specific marker. The cells of 2HP₁ are tumorigenic when tested in athymic mice, but the cells of 2HP are not. The establishment of amylase-producing human parotid adenoma cell lines of different characteristics in culture provides a new opportunity to study the mechanisms of differentiation and transformation, and regulation ofα-amylase in these cells.     

Intrinsic and extrinsic factors in estrogen action in human breast cancer: role of polyamines and pituitary factors

Although polyamines are important in regulating proliferation of mammalian cells, their role in hormone induction of cell growth has not been delineated. In the estradiol-responsive human breast cancer cell line, T-47D clone 11, estradiol (10(-10) M) was able to stimulate cell proliferation and the activity of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in the biosynthesis of polyamines. alpha-Difluoromethylornithine (DFMO), a specific inhibitor of ODC, blocked the estradiol-induced cell proliferation and ODC activity. Exogenous addition of putrescine, the natural product of ODC, rescued the inhibitory effect of DFMO. In addition, DFMO abolished the estradiol-induced growth of several other estrogen-responsive human breast cancer cell lines but did not affect the growth of hormone-independent cell lines. Further, a serum factor was found to be required for estradiol to exert its effect. To gain insight into the nature of this and possibly other extrinsic factors involved, the effect of estradiol on the proliferation of T-47D cells transplanted into athymic nude mouse was evaluated. In this in vivo system, estradiol alone produced only moderate growth of the human breast tumor. The simultaneous transplantation of a prolactin (PRL)- and growth hormone (GH)-secreting rat pituitary tumor or normal rat pituitary glands at a different site dramatically potentiated the effect of estradiol on the growth of the breast tumor xenograft. Purified PRL or GH were without effect, indicating that the active pituitary factor is neither PRL nor GH. Further, conditioned medium from rat pituitary tumor cells potentiated the mitogenic effect of estradiol on T-47D and several other estrogen receptor-positive human breast cancer cell lines in vitro under serum-free condition. In conclusion, we have identified both intrinsic (polyamines) and extrinsic (pituitary/serum) factors that are importance for estrogen to exert its mitogenic action. The next goal will be to elucidate the mechanisms of action of these molecules in the modulation of estrogen action.     

Observations consistent with autocrine stimulation of hybridoma cell growth and implications for large-scale antibody production

Existence of autocrine growth factors (aGFs) may influence the serum requirement for growth of hybridoma cells and thus significantly influence process economics. For the murine hybridoma cell line S3H5/2bA2, critical inoculum density (cID) and serum requirement for growth were inversely related for cultivation in both T flasks and spinner flasks. In spinner flasks, an inoculum density of 10⁶ cells/ml was necessary for the cells to grow in RPMI 1640 medium without serum supplement, and an inoculum density of 10³ cell/ml was necessary in RPMI 1640 medium with 10% serum. In T flasks, where the local cell density is higher than in spinner flasks, an inoculum density of 10⁶ cells/ml was necessary for the cells to grow in RPMI 1640 medium without serum supplement, and an inoculum density of 1 cell/ml was also necessary in RPMI 1640 medium with 10% serum. Further, immobilized cells at high local cell density could grow under conditions where cells in T flasks at corresponding overall cell density could not grow. The cells at high inoculum density were less sensitive to shear induced by mechanical agitation than the cells at low inoculum density. Taken together these observations support the existence of secreted aGF(s) by the hybridoma cell line used. Since the specific MAb production rate was independent of cultivation method and inoculum density, the existence of autocrine growth factors would suggest that the use of immobilized cells should improve the economics of MAb production.     

CCAAT/Enhancer binding protein delta (c/EBPdelta) regulation and expression in human mammary epithelial cells: I. "Loss of function" alterations in the c/EBPdelta growth inhibitory pathway in breast cancer cell lines

"Loss of function" alterations in growth inhibitory signal transduction pathways are common in cancer cells. In this study, we show that growth arrest (GA) treatments--serum and growth factor withdrawal and growth inhibitory IL-6 family cytokines (Interleukin-6 and Oncostatin M (OSM))--increase STAT3 phosphorylation (pSTAT3), increase CCAAT enhancer binding protein delta (C/EBPdelta) gene expression and induce GA of primary, finite-lifespan human mammary epithelial cells (HMECs), and immortalized breast cell lines (MCF-10A and MCF-12A). In contrast, serum and growth factor withdrawal from human breast cancer cell lines (MCF-7, SK-BR-3, T-47D, and MDA-MB-231) for up to 48 h induced a relatively modest increase in pSTAT3 levels and C/EBPdelta gene expression and resulted in varying levels of GA. In most breast cancer cell lines, IL-6 family cytokine treatment increased pSTAT3 levels and C/EBPdelta gene expression, however, growth inhibition was cell line dependent. In addition to "loss of function" alterations in growth inhibitory pathways, breast cancer cell lines also exhibit "gain of function" alterations in growth signaling pathways. The Akt growth/ survival pathway is constitutively activated in T-47D and MCF-7 breast cancer cells. The Akt inhibitor LY 294,002 significantly enhanced T-47D growth inhibition by serum and growth factor withdrawal or IL-6 family cytokine treatment. Finally, we show that activation of the pSTAT3/C/EBPdelta growth control pathway is independent of estrogen receptor status. These results demonstrate that "loss of function" alterations in the pSTAT3/C/EBPdelta growth inhibitory signal transduction pathway are relatively common in human breast cancer cell lines. Defective activation of the pSTAT3/ C/EBPdelta growth inhibitory signal transduction pathway, in conjunction with constitutive activation of the Akt growth stimulatory pathway, may play a synergistic role in the etiology or progression of breast cancer.     

A comparison of the effects of hyperthermia on cell growth in human T and B lymphoid cells: relationship to alterations in plasma membrane transport properties

Hyperthermic exposure (39-43 degrees C) for 1 or 2 hr impairs growth and Na+-dependent amino acid transport in both a radiosensitive human T (Molt-4) and a radioresistant B (RPMI 1788) lymphoid cell line. The heat damage to Na+-dependent amino acid transport in both cell lines is reversible under the conditions tested. Cell growth, as judged by increases in cell number, is decreased in both cell lines after hyperthermic treatment (43 degrees C, 1-hr exposure). This decrease in growth correlated with the damage to, and recovery of, the Na+-dependent amino acid transport system. However, the sensitivity to heat of both growth and Na+-dependent amino acid transport appears to differ in Molt-4 which is somewhat more sensitive to hyperthermia (T-cell line) vs RPMI-1788 (B-cell line). In the case of Molt-4, the rate of growth is decreased for about 60-80 hr after cells are exposed for 1 hr at 43 degrees C; whereas increases in cell number in the RPMI 1788 is observed within 40 hr after the heat treatment. The differences observed in cell growth and transport in these two lymphoid cell lines are attributed to the manner in which heat affects (i) the transport parameters in Molt-4 vs RPMI 1788 (i.e., the Michaelis-Menten constants Km and Vmax) and (ii) the putative plasma membrane sulfhydryl protein(s) which modulates Na+-dependent amino acid transport.     

Establishment and Characterization of Cell Lines from Homozygous Brachyury (T/T) Embryos of the Mouse

Lethal mutations in the T/t complex cause stage-specific morphologic abnormalities during early embryogenesis of mice. Although mutant embryos are lethal at the early stages of development, we have succeeded in establishing several cell lines from one of these mutants ( T/T ). Mutant-specific abnormality was not observed in gross morphology and growth patterns of cells. They, however, retained the characters of freshly dissociated embryonic cells to form smaller aggregates than the wild-type in rotation-mediated aggregation.
One of the T/T cell lines (T-1) formed tumors when injected into one-day-old syngeneic and allogeneic host, Expression of H-2 antigens was serologically studied with H-2 specificity 5 as a marker antigen. All lines except T-1 were shown to have this specificity.     

The cultivation of mouse and human lymphoid cells on serum-free media]

The optimum composition of several serum-free media has been established for a long-term cultivation of hybridomas, lymphoid and erythroleukemic cells. The medium DME/F12 appeared to be the medium of choice. It is necessary to supplement the basic medium with lipid and iron transport proteins (bovine serum albumin, transferrin) and peptide hormone (insulin) for obtaining stable results. However, there are differences in successful growth of examined cell lines under serum-free conditions: some of them acquire saturation density comparable with that of the control medium (hybridomas derived from myeloma Sp2/0-Ag14, cell lines K-562, Raji) but other lines do not (hybridoma derived from myeloma NS0/1, cell lines Namalwa, RPMI 1788, Molt-4). Thus, these serum-free media are not universal, therefore each new hybridoma and cell line should be tested to determine the suitability for them of some proposed media. The high effectiveness of cultivation under serum-free conditions can be presumably achieved by optimization of both qualitative and quantitative composition of the serum replacement and of the basic medium.     

Regulation of transforming growth factor alpha and transforming growth factor beta messenger ribonucleic acid abundance in T-47D, human breast cancer cells

Both transforming growth factor beta (TGF beta) and TGF alpha mRNA are expressed in human breast cancer cell lines. We have investigated the relationship of mRNA abundance for these growth modulators to the proliferation rate of a number of human breast cancer cell lines. Furthermore, we have investigated the relationship of regulation of TGF beta and TGF alpha mRNA to growth inhibition caused by progestins and nonsteroidal antiestrogens in T-47D human breast cancer cells. The abundance of TGF beta and TGF alpha mRNA in human breast cancer cell lines was not related directly to proliferation rate of the cells in culture or estrogen receptor positivity or negativity. The relationship of TGF beta and TGF alpha mRNA to growth inhibition caused by antiestrogens and progestins was investigated in T-47D human breast cancer cells. We observed that in T-47D human breast cancer cells the abundance of TGF beta mRNA is decreased in a time- and dose-dependent fashion by progestins but remains unaltered by nonsteroidal antiestrogens. Treatment of T-47D cells for 24 h with 10 nM medroxyprogesterone acetate (MPA) reduced the level of TGF beta mRNA to one third that present in untreated cells. The same treatment increased TGF alpha mRNA 3-fold above untreated controls in a time- and dose-dependent fashion and nonsteroidal antiestrogens caused a small decrease. The regulation of both TGF alpha and TGF beta mRNA was not directly related to inhibition of growth by progestins and antiestrogens in T-47D cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nonhaem complexes of FeIII stimulate cell attachment and growth by a mechanism different from that of serum, 2-oxocarboxylates, and haemproteins

Most cell lines, even those producing their own growth factors, need a serum supplement when growing in several commonly used media. The requirement for serum to sustain attachment and growth in RPMI 1640 and MEM has been found to be met by a range of 2-oxocarboxylates, by diverse coordination complexes of FeIII, and by a variety of haem-containing proteins including catalase. The latter directly implicates H2O2 in the serum shift-down effects. H2O2 was found to accumulate in low serum media under normal laboratory lighting conditions to levels that were shown to be sufficient, when added to freshly prepared media, to explain the depressed cell performance. With the exception of some of the nonhaem FeIII coordination complexes, substances found to stimulate cell attachment and growth were capable of scavenging H2O2. This suggests that an important function of serum and the 2-oxocarboxylates (alpha-keto acids) frequently used as "nonessential" medium additives is to remove H2O2 produced photodynamically during the storage and manipulation of media containing a high content of riboflavin. However, the nonhaem FeIII complexes with saturated coordination shells, although capable of reducing photodynamic generation of H2O2 to a greater or lesser extent, have their prime effect by an unknown, intriguing mechanism, probably based on a common redox function.     

Serum-free medium for murine and human lymphoid and hybridoma cell lines

We established a serum-free medium of low protein content(125g/ml) TYI 100, consisting of three hormones and five growth factors for the growth of lymphoid and hybridoma cell lines. In TYI 100 medium, mouse and human hybridomas grew equally well as in RPMI 1640 supplemented with 10% fetal bovine serum (10% FBS) without adaptation to the serum-free medium. TYI 100 medium allowed several passages of mouse hybridoma lines and the total cell number was more than in 10% FBS. TYI 100 medium also supported growth of myelomas and anchorage dependent cell lines, Bowes and CHO, well. TYI 100 medium is composed of inexpensive supplements and is therefor applicable to large scale culture.Abbreviations FBS Fetal Bovine Serum - IMDM Iscove's Modification of Dulbecco's Medium - PBS Phosphate-Buffered Saline - TPA Tissue Plasminogen Activator     

Glutamine-limited batch hybridoma growth and antibody production: experiment and model

The influence of glutamine (a major energy source) on both hybridoma growth and monoclonal antibody production was examined. A series of batch experiments were performed in T-flasks containing initial glutamine levels ranging from 0.5 to 4.0 mM in RPMI 1640 with 20% v/v fetal calf serum. The maximum final cell concentration increased with initial glutamine levels in the range of 0.5-2 mM; further glutamine increases had little or no effect. Earlier studies in our laboratories demonstrated that serum component(s) strongly influence the maximum specific growth rate. Here, the present studies reveal also the stoichiometric limitation by glutamine in the later stages of growth when its concentration is drastically reduced. For 0.5 to 1.5 mM initial glutamine, complete substrate utilization coincided with the cessation of cell growth and the onset of the death phase. For initial glutamine concentrations higher than 2.0 mM, growth halted prior to glutamine exhaustion, presumably because serum or RPMI component(s) were exhausted. The specific antibody secretion rate was essentially non-growth-associated above a critical low glutamine concentration in both the growth and death phases. At or below this critical value, an apparent emergence of stoichiometnc or energy limitation resulted in a dramatic drop in the secretion rate to zero. A simple unstructured model was developed that simulates these trends well. All parameters were determined using only subsets of the data. Nevertheless, these parameter values provided simulations in good agreement with all the glutamine-limited cultures.     

Estrogen-induced apoptosis in a breast cancer model resistant to long-term estrogen withdrawal

Estrogen suppression through the use of an aromatase inhibitor is an effective endocrine treatment option for postmenopausal breast cancer patients with estrogen receptor (ER)-positive disease, however, there are concerns that long-term estrogen deprivation will inevitably lead to resistance. To address the issue of acquired resistance to long-term estrogen deprivation our laboratory has developed an ER+/PR- hormone-independent breast cancer cell line, MCF-7:5C which is a variant clone of wild-type MCF-7 cells. Originally, these cells were cultured in estrogen-free MEM containing 5% charcoal-stripped calf serum and were found to be resistant to both estradiol (E(2)) and antiestrogens. Interestingly, a completely different phenomenon was observed when MCF-7:5C cells were cultured in phenol red-free RPMI 1640 medium containing 10% charcoal-stripped fetal bovine serum (SFS). Using DNA quantitation assays, we examined the effect of E(2) on the growth of MCF-7:5C cells under different media conditions. Our results showed that 10(-9)M E(2) caused a dramatic 90% reduction in the growth of MCF-7:5C cells cultured in RPMI medium containing 10% SFS but did not have any significant inhibitory effects on cells cultured in MEM media. Additional experiments were performed to determine whether the medium or the serum facilitated the inhibitory effects of E(2) and the results indicated that it was the serum. Annexin V and DAPI staining confirmed that the E(2)-induced growth inhibition of MCF-7:5C cells was due to apoptosis. We also examined the tumorigenic potential of MCF-7:5C cells by injecting 1x10(7)cells/site into ovariectomized athymic mice and found that these cells, previously cultured in RPMI media, spontaneously grew into tumors in the absence of E(2). Overall, these results show that low concentrations (>10(-11)M) of E(2) are capable of inducing apoptosis in an aromatase resistant breast cancer cell model and that this effect is highly influenced by the medium in which the cells are grown.     

Phosphorylcholine is favorable for antibody production from hybridoma cells

Growth of antibody-secreting hybridomas requires special conditions such as serum-free defined media containing growth factors and vitamins. However, the surface on which these cells can proliferate has been shown to play an important role. Phosphorylcholine (PC)-based polymers are zwitterionic compounds with nonbiofouling properties. These polymers are characterized by having reduced protein absorption properties. Our aim was to determine whether well-established hybridoma cell lines were able to proliferate and produce measurable amounts of monoclonal antibodies when grown on PC-polymer-coated surfaces. Comparative experiments using four well-known hybridoma cell lines (PAb421, PAb246, PAb1801 which recognize p53, and PAb280 which recognizes SV40 small t antigen) grown on PC-polymer-coated, uncoated, and two commercially available tissue culture plates showed that PC-polymer-coated plates were more efficient than uncoated plates in sustaining cell growth and monoclonal antibody production/secretion as defined by growth assays and ELISA. Also, results demonstrated that PC-polymer-coated plates were able to perform better than commercially available plates. These observations suggest that PC polymers could be used as an alternative, efficient surface coating to grow hybridoma cell lines and allow detectable antibody secretion.     

Morphological and proliferative characteristics of human breast tumor cells cultured on plastic and in collagen matrix

Summary Collagen, a major component of the extracellular matrix, is important in maintaining the in vivo characteristics of epidermal cells in vitro. In the present study, the morphological and proliferative characteristics of two human mammary epithelial cell lines (T-47D and MCF-7) cultured in cowhide collagen (Vitrogen 100) were studied. When grown in collagen, the tumor cells displayed a spherical shape and formed multilayered, tumorlike aggregates; desmosomes were observed between cells. In contrast, both cell lines grew as monolayers on plastic substratum; cells were characteristically flat and polygonal. When grown in collagen matrix, the human breast cancer cells became more dependent on serum for growth: cells proliferated in the presence of 10% fetal bovine serum (FBS) but failed to grow in 1% serum. On the other hand, these cells proliferated rapidly in 1% serum when they were grown on plastic. Even in 10% serum the doubling time of cells cultured in collagen was longer than that of cells maintained on plastic. In addition, cells cultured in collagen proliferated rapidly in a serum-free medium containing insulin, epidermal growth factor (EGF), estrogen, and transferrin. The collagen gel system may be useful for characterizing physiologically important trophic factors that regulate the proliferation and other functions of human breast tumor cells. The advice of Drs. J. A. Paterson and B. Dronzek in the electron microscopy studies is appreciated. This research was supported by the Medical Research Council of Canada. Clement K. H. Leung was supported by a University of Manitoba graduate fellowship. Portions of this work were reported at the Twentieth Annual Meeting of the American Society for Cell Biology held in Cincinnati, Ohio, November 14–18, 1980.     

Karyotypic variability of human multiple myeloma cell lines

Cytogenetic analysis of human multiple myeloma (MM) cell lines L363, Karpas 707, RPMI 8226, and U-266 was carried out. During long-term existence in vitro, the number of chromosomes in the cell lines was shown to be preserved at the near-diploid level (L363, Karpas 707, U-266) or to increase up to the hypotriploid level (RPMI 8226). There were complexly rearranged karyotypes with abnormalities of chromosomes of all pairs in all cell lines; however, no identical chromosomal translocations have been revealed. Loci of chromosomes involved in structural rearrangements in these cell lines often coincided with sites of DNA copy number imbalances characteristic for MM in vivo. Distinct types of the karyotypic structure of cell populations differing in the combination of cells with the main and additional structural variants of karyotype and of cells with nonclonal chromosome rearrangements were found in MM cell lines. In general, the karyotypic variability of the MM cell lines corresponds to the dynamics of karyotype of myeloma cells in vivo and, hence, has a tumor-specific character.     

Trichothecene-induced cytotoxicity on human cell lines

Trichothecene cytotoxicity of type A (T-2 toxin and HT-2 toxin), type B (deoxynivalenol, DON, and nivalenol, NIV), and type D (satratoxins G and H) compounds was determined comparatively by using eight permanent human cell lines (Hep-G2, A549, CaCo-2, HEp-2, A204, U937, RPMI 8226, and Jurkat). Viability of cells was measured by a water-soluble tetrazolium (WST-1) reagent cell proliferation assay assessing mitochondrial metabolic activity. Toxicity was expressed as the toxin concentration inhibiting 50% of cell viability (IC₅₀). Depending on the chemotype of the tested trichothecenes, relative cytotoxic activity differed by a factor of 100–1,000, and the corresponding IC₅₀ values were in the range from 2.2 nmol/l (satratoxin H on Jurkat and U937 cells) to 4,900 nmol/l (deoxynivalenol on HEp-2 cells). In contrast, the specific toxicity of each individual mycotoxin towards different cell lines was within remarkable close limits, and between-cell line differences were much smaller than previously reported. For the cell lines tested, IC₅₀ values were 4.4–10.8 nmol/l for T-2 toxin, 7.5–55.8 mol/l for HT-2 toxin, 600–4,900 nmol/l for DON, 300–2,600 nmol/l for NIV, and 2.2–18.3 nmol/l for satratoxins G/H. In addition, for the first time, the toxic activity of trichothecenes on primary cell culture of human endothelial cells (HUVEC) was tested. The susceptibility of this cell line was comparable to the other cell lines tested, with IC₅₀ values ranging from 16.5 nmol/l (T-2 toxin) to 4,500 nmol/l (DON). The results suggest that the current focus of cytotoxicological studies on trichothecenes on lymphoid cell lines may lead to an underestimate of their potential on other target cell systems.     

Evaluation of crude dextran as phase-forming polymer for the extraction of enzymes in aqueous two-phase systems in large scale

A detailed study of the influence of crude dextran on enzyme extractions in aqueous phase systems is presented in this article. The physical parameters of crude dextran, a purified T-500 fraction from Pharmacia, and a hydrolyzed crude dextran are compared and their influence on the phase system parameters investigated. Initially there is a drastic increase in the viscosity of the lower dextran-rich phase and a significant shift in the macroscopic structure of these phases, observed as the "gel-forming" properties of the dextran phases. The latter can be important for the partition of any enzyme by influencing the effect of phosphate concentration on the partition of proteins, although these experiments show that the partition coefficient of several enzymes is not much altered. The partition parameters allow the substitution of Dextran T-500 fractions by crude dextran or unfractionated, slightly hydrolyzed fractions. Using crude dextrans the performance and technical realization of enzyme extraction processes are demonstrated for pullulanase from Klebsiella pneumoniae and formate dehydrogenase from Candida boidinii.Both enzymes were recovered in comparable high yields. The equipment performance was quite good, as indicated by the high throughput values of the separators employed. Especially when using nozzle separators for phase separation there is a better performance in comparison to the Dextran T-500 fraction. No serious technical problems were encountered when replacing the expensive fractionated dextran with a crude dextran. In this way aqueous two-phase systems containing dextran become more feasible for enzyme purification from an economic point of view. The price of about 1.30 German marks (DM) per liter for a useful phase system already appears acceptable for the production of valuable intracellular enzymes.