Evaluation of a simple protein free medium that supports high levels of monoclonal antibody production

A simple protein free medium was formulated and tested in suspension culture using three hybridoma cell lines. The medium, referred to as CDSS (Chemically Defined Serum Substitutes), consisted of the basal medium DMEM:Ham F12, 1:1, with HEPES (D12H), plus pluronic F68, trace elements, ferric citrate, ascorbic acid, and ethanolamine. No protein or lipid components were added. All three cell lines were weaned off serum using CDSS and a commercially available protein free medium PFHM-II. Data shown here indicated that normally cells took 1–7 weeks to wean off serum and an additional 2–7 weeks to adapt to suspension culture. After adaptation the cells were able to grow well in suspension culture using both protein free media and in the main performed better than serum containing controls. The stability of the three hybridoma cells for antibody production following freeze/thaw procedures and long term subculturing was also tested. All three lines were frozen using our protein free CDSS medium (containing 0.75% bovine serum albumin and 10% dimethyl sulfoxide) in liquid nitrogen for up to one year. Cells thawed from these stocks recovered well and were able to maintain good growth and antibody production characteristics. One line was shown to grow using our protein free CDSS medium in suspension culture for 12 weeks without loss of antibody productivity.     

Development of a serum-free medium for dihydrofolate reductase-deficient chinese hamster ovary cells (DG44) using a statistical design: Beneficial effect of weaning of cells

Summary To develop serum-free (SF) medium for dihydrofolate reductase-deficient Chinese hamster ovary cells (DG44), a statistical optimization approach based on a Plackett-Burman design was adopted. DG44 cells which were normally maintained in 10% serum medium were gradually weaned to 0.5% serum medium to increase the probability of successful growth in SF medium. A basal medium was prepared by supplementing Dulbecco’s modified Eagle’s medium and Ham’s nutrient mixture F12 with hypoxanthine (10 mg/l) and thymidine (10 mg/l). Twenty-eight different supplements were selected as variables on the basis of their growth-promoting abilities. From statistical analysis, leucine, tryptophan, lysine, proline, histidine, hydrocortisone, ethanolamine, and phosphatidylcholine were identified as important components showing positive effects on cell growth. A new SF medium (SF-DG44) was formulated by supplementing the basal medium with these components. When the weaned cells were inoculated at 1.0 × 10⁵ cells/ml, a maximum viable cell concentration of 6.4×10⁵ cells/ml was achieved in SF-DG44 medium. In contrast, when the unweaned cells were used, a concentration of only 4.1×10⁵ cells/ml was reached under the same culture conditions, indicating that weaning of cells improves cell growth in SF medium. In summary, we found that development of a novel SF medium for DG44 cells was facilitated using a Plackett-Burman design technique and weaning of cells.     

Effects of peptone on hybridoma growth and monoclonal antibody formation

Hybridoma WuT3 secreting a monoclonal antibody against T lymphocytes was grown in RPMI 1640 medium supplemented with 1% human serum. The effect of the concentration of peptone, as an additive, was investigated on cell growth, monoclonal antibody formation, and cell metabolism over 0–10 g l^–1 range. It was found that 1–5 g l^–1 peptone can significantly promote the growth of cells and increase the formation of monoclonal antibody, especially at 3–5 g l^–1, when both the accumulating level and secretion rate of monoclonal antibody are higher than that at other peptone concentrations. Based on glucose, lactate and ammonia analysis data, the efficiency of glycolysis was assessed and the utilization of amino acids was more efficient at 3–5 g l^–1 peptone. The cell growth and monoclonal antibody formation were inhibited at higher peptone concentrations, e.g. 10 g l^–1.     

Antibody production and growth of mouse hybridoma cells in Nutridoma media supplements

Traditionally, cell culturists have relied upon the addition of serum to culture medium for the growth and maintenance of cell lines. However, many aspects of the use of serum in tissue culture are problematic. Cell culture supplements that circumvent the need for serum are readily available and provide a consistent protein composition. This defined environment allows the antibody to be more easily purified from culture supernatants. Nutridoma media supplements were formulated to support the growth of lymphoblastoid cells in a defined culture environment. In this study, Nutridoma media supplements were tested in parallel with serum-containing cultures to determine if Nutridoma supplemented medium is effective in supporting hybridoma cell growth and antibody production in three hybridoma cell lines. Data, based on cell growth and antibody production, show the importance of basal media selection when serum is replaced with Nutridoma media supplements. SDS-PAGE results show that cell supernatants from Nutridoma supplemented cultures contain very few contaminating proteins.     

Development of a serum-free culture medium for the large scale production of recombinant protein from a Chinese hamster ovary cell line

A serum-free medium, WCM5, has been developed for the large scale propagation of CHO (Chinese hamster ovary) cells which express recombinant protein using dihydrofolate reductase as a selectable marker. WCM5 was prepared by supplementing Iscoves medium without lecithin, albumin or transferrin with a number of components which were shown to benefit growth. WCM5 medium contained 5 mg l^–1 human recombinant insulin (Nucellin) but was otherwise protein-free. CHO 3D11^* cells which had been engineered to express a humanised antibody, CAMPATH^*-1H, were routinely grown using serum-containing medium. From a seeding density of 10⁵ cells ml^–1, cells grown in static culture with serum reached a maximal cell density of 6.5×10⁵ cells ml^–1 after 6 days in culture and produced a maximal antibody concentration of 69 mg l^–1 after 11 days in culture. CHO 3D11^* cells grown with serum were washed in serum-free medium then cultured in WCM5 medium. Following a period of adaptation the cell growth and product yield was superior to that achieved with serum-containing medium. CHO cells producing CAMPATH-1H grown in an 8000 l stirred bioreactor seeded with 2×10⁵ cells ml^–1 reached a maximal viable cell density of 2.16×10⁶ cells ml^–1 after 108 h in culture and a maximal antibody concentration of 131.1 mg l^–1 after 122 h in culture.Abbreviations CHO Chinese hamster ovary - dhfr dihydrofolate reductase - dhfr dihydrofolate reductase deficient - MTX methotrexate - H hypoxanthine - T thymidine - T/V trypsin versene - F12 Hams F12 medium - NEAA non essential amino acids     

Impact of Weaning from Acute Dialytic Therapy on Outcomes of Chronic Kidney Disease following Urgent-Start Dialysis

Discontinuation of acute, unplanned dialysis is always an important therapeutic goal in dialysis-requiring patients with existing chronic kidney disease. Only a limited proportion of patients could be weaned off dialysis and remained dialysis-free. Here we performed a multicenter, observational study to investigate factors associated with successful weaning from acute dialysis, and to explore the potential impact of weaning itself on outcomes of patients with chronic kidney disease following urgent-start dialysis. We recruited 440 chronic kidney disease patients with a baseline estimated glomerular filtration rate <45 ml/min per 1/73 m2, and used propensity score-adjusted Cox regression analysis to measure the effect of weaning from acute dialysis on death during the index hospitalization and death or readmission after discharge. Over 2 years, 64 of 421 (15.2%) patients who survived >1 month died, and 36 (8.6%) were removed from dialysis, with 26 (6.2%) remaining alive and dialysis-free. Logistic regression analysis found that age ≧ 65 years, ischemic acute tubular necrosis, nephrotoxic exposure, urinary obstruction, and higher predialysis estimated glomerular filtration rate and serum hemoglobin were predictors of weaning off dialysis. After adjustment for propensity scores for dialysis weaning, Cox proportional hazards models showed successful weaning from dialysis (adjusted hazard ratio 0.06; 95% confidence interval 0.01 to 0.35), along with a history of hypertension and serum albumin, were independent protectors for early death. Conversely, a history of stroke, peripheral arterial disease and cancer predicted the occurrence of early mortality. In conclusion, this prospective cohort study shows that compared to patients with chronic kidney disease who became end-stage renal disease after acute dialysis, patients who could be weaned off acute dialytic therapy were associated with reduced risk of premature death over a 2-year observation period.     

Physiological changes during the adaptation of hybridoma cells to low serum and serum-free media

Two murine hybridoma cell lines (167.4G5.3 and S3H5/gamma2bA2) were adapted to grow in low-serum and serum-free media by a weaning procedure. The changes in cell growth, metabolic, and antibody production rates with adaptation were examined using biochemical and flow cytometric analyses. After adaptation to a particular serum level, the short-term serum response of the cells was experimentally determined. Specific growth rates, glucose and glutamine uptake and lactate and ammonia production rates, and specific antibody production rates were evaluated from the data. For both cell lines, an improvement in cell growth was observed after adaptation, and both higher growth rates and higher cell concentrations were obtained. The specific glucose and glutamine uptake rates and the lactate and ammonia production rates changed insignificantly with adaptation. Conversely, changes in the specific antibody production rate of the two cell lines differed. Cell line 167.4G5.3 showed a loss in antibody productivity at low serum levels, while the S3H5/gamma2bA2 kept its original productivity in low-serum-containing media. The intracellular antibody content for S3H5/gamma2bA2 cells remained unaltered by adaptation, but a low antibody containing cell population appeared in the 167.4G5.3 culture. The loss of specific antibody productivity in this cell line was due to the appearance of this population.     

Hybridoma growth in a new generation hollow fibre bioreactor: antibody productivity and consistency

This paper analyses the performance of MAbMax^TM/Tricentric^TM, a new generation hollow fibre bioreactor, for hybridoma growth and antibody productivity, the down stream processing of monoclonal antibody harvests throughout the run and the further control of antibody quality consistency. Handling and process parameters were optimised using a mouse hybridoma, IgG1_K secretor, and then confirmed with several other hybridomas. Cells were kept at optimal viability during an unusually long period of time and a continuously high production of antibodies was detected over several months. Foetal bovine serum concentration was reduced to 1\% and the effects of weaning of cells from serum were monitored in terms of cell metabolism and antibody productivity. Antibody harvests collected at regular intervals throughout the run (2 to 12 weeks) were purified using affinity chromatography on a recombinant protein A/G matrix and then analysed in terms of antigen binding properties, isoelectric forms and oligosaccharide structures, in order 1) to control antibody quality consistency as a function of time and serum concentration and 2) to compare antibody characteristics as a function of culture conditions, in vitro bioreactor cultivation versus in vivo mouse ascite cultivation. This revised version was published online in July 2006 with corrections to the Cover Date.     

复合活菌制剂对断奶仔猪生长性能及血清溶菌酶含量的影响

目的观察复合活菌制剂对断奶仔猪生长性能及血清溶菌酶含量的影响。方法选择长白二元杂交断奶仔猪90头进行实验,断奶日龄为35 d。共分为5个组,每组设3个重复,每个重复随机选取健康仔猪6头。实验组一:饲喂基础日粮+0.1%复合制剂(纳豆杆菌,双歧杆菌,罗伊乳杆菌),实验组二:饲喂基础日粮+0.1%复合制剂(纳豆杆菌,双歧杆菌,干酪乳杆菌),实验组三:饲喂基础日粮+0.1%复合制剂(纳豆杆菌,双歧杆菌,嗜酸乳杆菌),实验组四:饲喂基础日粮+0.1%复合制剂(纳豆杆菌,双歧杆菌,罗伊乳杆菌,干酪乳杆菌,嗜酸乳杆菌),对照组:饲喂基础日粮。其中复合制剂中益生菌活菌数为109CFU/g。饲养30 d后观察复合活菌制剂对断奶仔猪生长性能及血清溶菌酶含量的影响。结果在日增重、饲料效率及血清溶菌酶方面,实验组一和二显著高于对照组(P0.05);在腹泻率方面,实验组均显著低于对照组(P0.05)。结论在仔猪日粮中添加复合活菌制剂可提高每头断奶仔猪平均日增重及饲料效率,降低腹泻的发病率,且增高仔猪血清溶菌酶含量,提高仔猪的免疫机能,从而提高经济效益。     

High frequency plant regeneration from zygotic-embryo-derived embryogenic cell suspension cultures of watershield (Brasenia schreberi)

An improved protocol for high frequency plant regeneration via somatic embryogenesis from zygotic embryo-derived cell suspension cultures of watershield (Brasenia schreberi) was developed. Zygotic embryos formed pale-yellow globular structures and white friable callus at a frequency of 80% when cultured on half-strength MS medium supplemented with 0.3 mg l⁻¹ 2,4-D. However, the frequency of formation of pale-yellow globular structures and white friable callus decreased slightly with increasing concentrations of 2,4-D up to 3 mg l⁻¹, where the frequency reached ~50% of the control. Cell suspension cultures from zygotic embryo-derived white friable callus were established using half-strength MS medium supplemented with 0.3 mg l⁻¹ 2,4-D. Upon plating of cell aggregates on half-strength MS basal medium, approximately 8.3% gave rise to somatic embryos and developed into plantlets. However, the frequency of plantlet development from cell aggregates was sharply increased (by up to 55%) when activated charcoal and zeatin were applied. Regenerated plantlets were successfully transplanted to potting soil and grown to normal plants in a growth chamber. The distinctive feature of this study is the establishment of a high frequency plant regeneration system via somatic embryogenesis from zygotic embryo-derived cell suspension cultures of watershield, which has not been previously reported. The protocol for plant regeneration of watershield through somatic embryogenesis could be useful for the mass propagation and transformation of selected elite lines.     

Production of rat monoclonal antibody from rat x mouse hybridoma cell lines using microencapsulation technology

Summary The cell growth and monoclonal antibody production characteristics of two rat x mouse heterohybridoma cell lines, designated 187.1 and M1/9.3, were investigated using a biocompatible microencapsulation technology. Both cell lines, derived from the fusion of immunized rat spleen cells with either the NS1 or X63Ag8.653 myeloma cell lines, were found to reach a maximum intracapsular cell density of 1.3 to 1.5×10⁷ cells/ml during a 27-d culture period. During this period, rat monoclonal antibody accumulated in the intracapsular space of both cultures to a final concentration of 2.0 to 2.8 mg/ml. Comparison of the concentration of rat monoclonal antibody in the extracapsular vs. the intracapsular space of both cultures indicated that significantly less than 1% of the antibody produced by the encapsulated hybridoma cells was capable of transiting the microcapsule membrane during the culture period. Due to the partition of the rat monoclonal antibody within the intracapsular space, the initial purity of the antibody harvested from 21-d microcapsule cultures of 187.1 and M1/9.3 cells was approximately 48 and 75% by weight, respectively. Analysis of the intracapsular protein by sodium dodecyl sulfoxide gel electrophoresis at different times during the culture period demonstrated that the principal contaminant associated with the unpurified antibody was bovine serum albumin.     

Flow cytometric analysis of experimental parameters for the immunofluorescent labeling of BrdUrd in various tumour cell lines

Summary This report describes the results of the comparison of three different methods and three monoclonal antibodies to stain cells in suspension for incorporated bromodeoxyuridine and total DNA content. The procedures were tested in three different experimental tumour cell lines. The sensitivity of the different procedures was expressed as the ratio of the anti-BrdUrd fluorescence intensities of the S and G1 phase cells (FS/FG1 ratio). There were remarkable differences in sensitivity between the different procedures. With the heat denaturation the most favourable FS/FG1 ratio's were obtained but substantial cell loss occurred during this procedure which is a disadvantage for clinical application. With the pepsin digestion + acid denaturation procedure cell loss was negligible. The standard acid denaturation procedure was inferior to the other two methods. Using the pepsin digestion + acid denaturation procedure we examined the variations in sensitivity for the different monoclonal antibodies and cell lines and the influence of BrdUrd concentration, labelingtime and cell concentration. The binding characteristics for the various antibodies differed considerably in our hands. Only with the IU4 antibody we obtained FS/FG1 ratio's comparable with those desenbed in the literature. No difference was observed between the cell lines. Variation in cell concentration between 1 × 10⁴ to 1 × 10⁶ ml nor BrdUrd concentration appeared to influence the sensitivity of the procedure. A labelingtime of 1 h or even 30 min seems to be more than sufficient for an optimal FS/FG1 ratio.Our results indicate that using the appropriate antibody and immunofluorescence BrdUrd can be detected by flow cytometry, after incorporation into the DNA of tumour cells under a wide range of culture conditions.For clinical application, the pepsin digestion + acid dena uration method in combination with IU4 antibody seems to be the procedure of choice due to its good reproducibility, sensitivity and its low cell loss.     

In vitro clonal propagation and stable cryopreservation system for <Emphasis Type="Italic">Platycladus orientalis</Emphasis> via somatic embryogenesis

Platycladus orientalis is a widespread conifer, which is native in eastern Asia, and has recently attracted much attention due to its ornamental value for landscape and gardens. However, native P. orientalis populations have been in decline over the past century. Here, we established an in vitro propagation and cryopreservation system for P. orientalis via somatic embryogenesis (SE). Whole megagametophytes with four development stages (Early embryogeny: E1 and late embryogeny: L1, L2, and L3) of zygotic embryos from immature P. orientalis cones were used as initial explants and cultured on three different basal media such as initiation medium (IM), Litvay (LV), and Schenk and Hildebrandt (SH). Both the developmental stage of zygotic embryos and kind of basal medium had a significant effect on embryogenesis induction with IM (P?<?0.001, respectively). The highest frequency of embryogenic callus induction was obtained in megagametophytes with zygotic embryos at L2 stage, which ranged as high as 30%. The maturation medium containing IM basal salts, vitamins and amino acids, 15 g l^?1 abscisic acid (ABA), 50 g l^?1 maltose, and 100 g l^?1 polyethylene glycol 4000 (PEG) was found to be the suitable medium for production of somatic embryos. The frequency of somatic embryo formation from both non-cryopreserved and cryopreserved cell lines was also tested. There were no statistical differences on the production of somatic embryos between non-cryopreserved and cryopreserved cells (P?=?0.523). Genetic fidelity of the plantlets regenerated from non-cryopreserved and cryopreserved embryogenic cell lines was assessed by both random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) analysis. There was no genetic instability in the regenerated plantlets from cryopreserved embryogenic cell lines. Both the SE protocol and cryopreservation protocols described here have the potential to contribute the conservation and clonal propagation of P. orientalis germplasm.     

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.     

A protocol to effectively create single cell suspensions of adherent cells for multiparameter high-throughput flow cytometry

High-throughput flow cytometry of adherent cells is difficult because the creation of single cell suspensions can damage cells and yield artificial results. We describe a protocol to increase the single cell suspension yield of adherent human cells without injury. Doxorubicin, a cytotoxic agent, was administered to adherent human pancreatic carcinoma cell lines (Panc-1 and AsPC-1) to produce alterations in the cell cycle and intracellular protein expression. The cells in 96-well plates were disassociated using a collagenase and trypsin mixture. Fluorescence-activated high-throughput flow cytometry evaluated cellular viability as well as surface and intracellular protein expression. Cell cycle analysis was performed using 7-aminoactinomycin D and intracellular protein characterization was performed using a fluorescein-labeled monoclonal antibody against activated caspase-3. The collagenase–trypsin-based protocol increased single cell events from 31.9 ± 0.5% using trypsin alone (standard) to a range of 62.1% to 85.5% without adversely affecting viability. High-throughput flow cytometry demonstrated that the addition of collagenase to the disassociation solution not only permitted significantly higher rates of single cell creation, but it did not negatively affect the doxorubicin-induced protein expression. This protocol allows for expedient and effective disassociation of adherent human cells in order to investigate alterations in specific cellular enzymes and pathways.     

The use of serum-free medium for the production of functionally active humanised monoclonal antibody from NS0 mouse myeloma cells engineered using glutamine synthetase as a selectable marker

A protein-free growth medium (W38 medium) had previously been developed for the NS0 mouse myeloma cell line which is cholesterol-auxotrophic. This paper describes the development of a protein-free growth medium for NS0 cells expressing humanised monoclonal antibody using GS (glutamine synthetase) as a selectable marker. Several GS-engineered NS0 cell lines expressing humanised monoclonal antibody grew in a modification of W38 medium which maintained GS-selection, supplemented with cholesterol, phosphatidylcholine and -cyclodextrin. Further studies showed that additional glutamic acid, asparagine, ribonucleosides and choline chloride improved cell growth. Amino acid analysis identified a number of amino acids that were being depleted from the culture medium. NS0 cell lines 9D4 and 2H5 expressing CAMPATH-1H^* were adapted to enable them to grow serum-free in the absence of cholesterol and -cyclodextrin. Cholesterol-independent 9D4 (9D4.CF) cells grown in shake flask culture using an enriched protein-free medium (WNSD medium), supplemented with human recombinant insulin (Nucellin), reached a maximum cell density to 1.86×10⁶ cells ml^–1 producing 76.6 mg l^–1 of antibody. CAMPATH-1H antibody produced using serum-free medium was found to be functionally activein vitro in the Antibody Dependant Cellular Cytotoxicity (ADCC) assay.Abbreviations C cholesterol - CD cyclodextrin - dhfr dihydrofolate reductase - F68 Pluronic F68 - GS glutamine synthetase - MSX methionine sulphoximine - P phosphatidylcholine - PC-FBS phosphatidylcholine, cholesterol and foetal bovine serum - RPMI RPMI 1640 medium - ADCC Antibody-dependant cellular cytotoxicity     

Neuraminidase reversal of resistance to lysis of herpes simplex virus-infected cells by antibody and complement.

The susceptibility to lysis by antibody and complement was examined in four human cell lines. The cells were infected with herpes simplex virus type 1 and lysis was assessed by the 51Cr release test by using antibodies to herpes simplex virus and guinea pig serum as a source of complement. The four cell lines were found to differ in their susceptibility to lysis, although virus replication was readily demonstrated in the different cell lines. By indirect immunofluorescence, no differences in the expression of virus antigens at the surface of the cells could be found between the different cell lines. Treatment of cells with neuraminidase markedly enhanced the sensitivity of the cells which were relatively insensitive to lysis. The enhancement of susceptibiltiy to lysis by neuraminidase occurred if cells were treated before reaction of the cells with antibody and if the cells were reacted with antibody before treatment with the enzyme. No enhancement was observed when cells were reacted with antibody and complement before neuraminidase treatment. Neuraminidase treatment did not seem to enhance appreciably the quantity of antibody which reacted at the cell surface. The observations suggest that surface properties of certain cells render the cells resistant to lysis by antibody and complement and that the resistance to lysis can be abrogated by treating the cells with neuraminidase.     

A novel feeding strategy for enhanced protein production by fed-batch fermentation in recombinant Pichia pastoris

A novel feeding strategy for enhanced protein production of hepatitis B virus surface antigen (HBsAg) in fed-batch fermentation, recombinant Pichia pastoris, has been developed. A minimal salt medium was used to grow cells in the initial batch fermentation, followed by a glycerol+salts fed-batch phase. At the end of the fed-batch phase a dry cell weight of 130 g l⁻¹ was achieved. In the absence of basal salts, the same amount of glycerol feed resulted in only 90 g l⁻¹ cell dry weight. When a limited amount of casamino acids were also included every 24 h during methanol induction, there was a two-fold increase in expression levels of HBsAg. After 192 h of induction, the expression levels of HBsAg (soluble and insoluble) reached >1 g l⁻¹ using the Mut⁻ strain. Thus, the use of basal salts in the glycerol feed, along with the addition of limited amounts of casamino acids with the methanol feed, resulted in an increased expression of total HBsAg.     

Chickpea protein hydrolysate as a substitute for serum in cell culture

The growth of mammalian cells in vitro requires the use of rich culture media that are prepared by combining serum with specific nutrient formulations. Serum, the most expensive component of culture media, provides a complex mixture of growth factors and nutrients. Protein hydrolysates that can support in vitro cell growth and eliminate or reduce the need to use serum have been obtained from different sources. Here we describe the use of two food grade proteases to produce a chickpea protein hydrolysate that has been added to cell culture medium in order to determine whether it can be used as a substitute for serum. Medium containing the hydrolysate has been tested using two human cells lines: the monocytic THP-1 cell line which grows in suspension, and the epithelial Caco-2 cell line which grows as a monolayer. The chickpea protein hydrolysate was a good substitute for serum in the first case, but did not allow growth of Caco-2 cells. Supplementation of culture media with this inexpensive and safe hydrolysate would greatly reduce the cost of cell culture.     

Pemphigoid, pemphigus and desmoplakin as antigenic markers of differentiation in normal and tumorigenic mouse keratinocyte lines

Abstract The expression of differentiation stages in a murine epidermal cell transformation model has been investigated as a basis for studies of chemically-induced differentiation. Antibodies in sera of patients with the autoimmune diseases bullous pemphigoid and pemphigus vulgaris exhibit specific reactivity to antigenic determinants of basal and spinous cells, respectively, in sections of mouse and human epidermis. In addition, spinous cells in epidermis are reactive with a mouse monoclonal antibody to desmoplakin, a desmosomal component immunologically distinct from pemphigus. These antibodies were used to identify and attempt to quantify keratinocyte subpopulations in culture based on differentiation stage. Epidermal cell lines were cultured under conditions which favour proliferation (0.02 to 0.04 mm extracellular Ca²⁺, i.e. low Ca²⁺ conditions) or differentiation (0.1 mM to 1.4 mM Ca²⁺), as previously shown using primary cultures of mouse keratinocytes. Two independently-derived normal keratinocyte lines demonstrated Ca²⁺-dependent reactivity with pemphigoid and pemphigus antiserum, like that which has been observed in primary cultures. Furthermore, a Ca²⁺ and time-dependent reactivity with the three antisera was also observed in a papilloma cell line (derived from one of the normal cell lines after treatment in vitro with 7,12-dimethylbenz[α]anthracene). Papilloma cells cultured under conditions of low extracellular Ca²⁺ were comprised of three subpopulations: cells reactive only with pemphigoid anti-serum, cells reactive with pemphigoid and desmoplakin antibody (intracellular location), and cells reactive only with desmoplakin antibody. However, like the normal cell lines, papilloma cells underwent a transition to predominantly a spinous cell population (i.e. reactive with pemphigus and desmoplakin antibody) in response to extracellular Ca²⁺. A slower loss of pemphigoid antibody reactivity was noted in papilloma cells, consistent with an abnormal regulation of differentiation. The attempt to characterize these dynamic transitions from basal to spinous cell subpopulations in culture was considered to be prerequisite for the use of the model to investigate differentiation-inducing agents in carcinoma therapy.