Short-term Primary Cell Cultures Research Articles

Human organoid systems in modeling reproductive tissue development, function, and disease.

Research focused on human reproductive biology has primarily relied upon clinical samples affording mainly descriptive studies with limited implementation of functional or mechanistic understanding. More importantly, restricted access to human embryonic material has necessitated the use of animals, primarily rats and mice, and short-term primary cell cultures derived from human patient material. While reproductive developmental processes are generally conserved across mammals, specific features unique to human reproduction have resulted in the development of human-based in vitro systems designed to retain or recapitulate key molecular and cellular processes important in humans. Of note, major advances in 3D epithelial stem cell-based systems modeling human reproductive organ development have been made. These cultures, broadly referred to as organoids, enable research aimed at understanding cellular hierarchies and processes controlling cellular differentiation and function. Moreover, organoids allow the pre-clinical testing of pharmacological substances, both from safety and efficacy standpoints, and hold large potential in driving aspects of personalized medicine that were previously not possible with traditional models. In this mini-review, we focus on summarizing the current state of regenerative organoid culture systems of the female and male reproductive tracts that model organ development, maintenance, and function. Specifically, we will introduce stem cell-based organoid models of the ovary/fallopian tube, endometrium, cervix, prostate gland, and testes. We will also describe organoid systems of the pre-implanting blastocyst and trophoblast, as the blastocyst and its extraembryonic trophectoderm are central to fetal, maternal, and overall pregnancy health. We describe the foundational studies leading to their development and outline the utility as well as specific limitations that are unique and common to many of these in vitro platforms.

Lymphoma and Leukemia Cell Vulnerabilities and Resistance Identified by Compound Library Screens.

Response to anticancer agents is often restricted to subsets of patients. The recognition of factors underlying this heterogeneity and the identification of biomarkers associated with response to drugs would greatly improve the efficacy of drug treatment. Platforms that can comprehensively map drug response in high-throughput ex vivo provide a unique tool to identify associated biomarkers and provide hypotheses for mechanisms underlying variable response. Such screens can be performed on cell lines and short-term cultures of primary cells to take advantage of the respective models' strength, which include, e.g., the ability to silence genes in cell lines and the "indefinite" supply of primary cells where clonal selection can be avoided. Cohorts of such samples represent the natural diversity of cancers, including rarer mutations and combinatorial patterns of mutations.We here summarize a simple and scalable method for the measurement of viability after drug exposure based on ATP measurements as a surrogate for viability, which we use to measure and understand drug response in cell lines and primary cells.

Metastasis-associated MCL1 and P16 copy number alterations dictate resistance to vemurafenib in a BRAFV600E patient-derived papillary thyroid carcinoma preclinical model.

BRAF(V600E) mutation exerts an essential oncogenic function in many tumors, including papillary thyroid carcinoma (PTC). Although BRAF(V600E) inhibitors are available, lack of response has been frequently observed. To study the mechanism underlying intrinsic resistance to the mutant BRAF(V600E) selective inhibitor vemurafenib, we established short-term primary cell cultures of human metastatic/recurrent BRAF(V600E)-PTC, intrathyroidal BRAF(V600E)-PTC, and normal thyroid (NT). We also generated an early intervention model of human BRAF(V600E)-PTC orthotopic mouse. We find that metastatic BRAF(V600E)-PTC cells elicit paracrine-signaling which trigger migration of pericytes, blood endothelial cells and lymphatic endothelial cells as compared to BRAF(WT)-PTC cells, and show a higher rate of invasion. We further show that vemurafenib therapy significantly suppresses these aberrant functions in non-metastatic BRAF(V600E)-PTC cells but lesser in metastatic BRAF(V600E)-PTC cells as compared to vehicle treatment. These results concur with similar folds of down-regulation of tumor microenvironment-associated pro-metastatic molecules, with no effects in BRAF(WT)-PTC and NT cells. Our early intervention preclinical trial shows that vemurafenib delays tumor growth in the orthotopic BRAF(WT/V600E)-PTC mice. Importantly, we identify high copy number gain of MCL1 (chromosome 1q) and loss of CDKN2A (P16, chromosome 9p) in metastatic BRAF(V600E)-PTC cells which are associated with resistance to vemurafenib treatment. Critically, we demonstrate that combined vemurafenib therapy with BCL2/MCL1 inhibitor increases metastatic BRAF(V600E)-PTC cell death and ameliorates response to vemurafenib treatment as compared to single agent treatment. In conclusion, short-term PTC and NT cultures offer a predictive model for evaluating therapeutic response in patients with PTC. Our PTC pre-clinical model suggests that combined targeted therapy might be an important therapeutic strategy for metastatic and refractory BRAF(V600E)-positive PTC.

Sensitivity of endometrial cancer cells from primary human tumor samples to new potential anticancer peptide lactaptin.

Endometrial carcinoma is the most common gynecologic malignancy which is associated with a poor prognosis when diagnosed at an advanced stage; therefore, the discovery of efficacious new drugs is required to reinforce conventional chemotherapy. Short-term cultures of primary cells from endometrial tumors could be used for testing new anticancer therapeutics as well as for the development of personalized cancer therapy strategy. Here, the antitumor effect of a recombinant analogue of lactaptin (RL2), a new potential anticancer molecule, was examined against primary human endometrial cancer cells. Primary cell cultures of malignant and normal human endometrium were performed by enzymatic digestion of endometrial tissue from biopsy material. Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed to determine the messenger ribonucleic acid (mRNA) state of estrogen (ERs) and progesterone (PRs) hormone receptors and aromatase (Cyp 19) in cell cultures. Dynamic monitoring of cell adhesion and proliferation was made using the iCELLigence system (ASEA Biosciences). The sensitivity of cell cultures to conventional anticancer drugs and the lactaptin analog was estimated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, flow cytometry, and the iCELLligence system. Established short-term primary cultures of endometrial cancer cells were ERα/ERβ/PR-positive and sensitive for RL2. The IC 50 values of doxorubicin and cisplatin were determined for all of the primary cultures designed. KE normal cells displaying low Cyp19 mRNA levels and high ERβ and PR mRNA levels were more resistant to RL2 treatment as well as to cisplatin and doxorubicin. Our results indicate that the recombinant analog of lactaptin, RL2, exerts cytotoxic effects against primary hormone-dependent endometrial tumor cells in vitro with features of apoptosis.

A Collection of Primary Tissue Cultures of Tumors from Vacuum Packed and Cooled Surgical Specimens: A Feasibility Study

Primary cultures represent an invaluable tool to set up functional experimental conditions; however, creation of tissue cultures from solid tumors is troublesome and often unproductive. Several features can affect the success rate of primary cultures, including technical issues from pre-analytical procedures employed in surgical theaters and pathology laboratories. We have recently introduced a new method of collection, transfer, and preservation of surgical specimens that requires immediate vacuum sealing of excised specimens at surgical theaters, followed by time-controlled transferring at 4°C to the pathology laboratory. Here we investigate the feasibility and performance of short-term primary cell cultures derived from vacuum packed and cooled (VPAC) preserved tissues. Tissue fragments were sampled from 52 surgical specimens of tumors larger than 2 cm for which surgical and VPAC times (the latter corresponding to cold ischemia time) were recorded. Cell viability was determined by trypan blue dye-exclusion assay and hematoxylin and eosin and immunohistochemical stainings were performed to appreciate morphological and immunophenotypical features of cultured cells. Cell viability showed a range of 84–100% in 44 out of 52 (85%) VPAC preserved tissues. Length of both surgical and VPAC times affected cell viability: the critical surgical time was set around 1 hour and 30 minutes, while cells preserved a good viability when kept for about 24 hours of vacuum at 4°C. Cells were maintained in culture for at least three passages. Immunocytochemistry confirmed the phenotype of distinct populations, that is, expression of cytokeratins in epithelioid cells and of vimentin in spindle cells. Our results suggest that VPAC preserved tissues may represent a reliable source for creation of primary cell cultures and that a careful monitoring of surgical and cold ischemia times fosters a good performance of primary tissue cultures.

The Molecular Basis for BCL-2 Oncogene Addiction in CLL.

CLL cells consistently express BCL-2 at a high level. Using a compound demonstrated to antagonize BCL-2 function, ABT-737, we show that CLL cells in short-term primary culture are uniformly sensitive to single-agent BCL-2 antagonism, with EC50's in the 10 nM range. To understand the mechanism of CLL sensitivity to this compound, we studied mitochondria from these primary CLL cells. We employed a panel of peptides derived from the BH3 domain of pro-death BH3-only proteins, certain of which selectively inhibit BCL-2 function in vitro. We demonstrate that those with activity against BCL-2 in vitro induce cytochrome c release, a hallmark of the mitochondrial dysfunction during apoptosis. A non-malignant cellular model of BCL-2 dependence revealed that binding of the pro-apoptotic protein BIM to BCL-2 correlated with sensitivity to BCL-2 inhibitors. We likewise discovered that BCL-2 binds and sequesters BIM in CLL cells. In contrast to the BCL-2 dependent CLL cells, we examined myeloma cell lines with defined MCL-1 dependence and relative BCL-2 independence. These cell lines were 100 fold less sensitive to ABT-737. Furthermore, in mitochondrial assays only BH3 peptides which interact with MCL-1 induce cytochrome c release. These experiments reveal that cellular requirement for an anti-apoptotic protein function can be deduced from a functional mitochondrial requirement determined using our panel of BH3 peptides. In CLL, and perhaps other malignancies, it appears that BCL-2 maintains survival by sequestering pro-apoptotic proteins like BIM. Inhibition of this sequestration provokes cell death ex vivo, even with a single agent BCL-2 inhibitor. Reliance on BCL-2 for survival may be an Achilles heel for CLL and other cancers.

Gefitinib is effective against juvenile pilocytic astrocytoma in vitro

Juvenile pilocytic astrocytomas (JPAs) are the most common central nervous system tumors in children. If completely resected, JPAs are associated with an excellent outcome. However, there is need for additional therapeutic approaches for those JPAs which are incompletely resected and fail subsequent standard chemotherapy/radiation. To explore the possibility for a novel therapeutic approach we measured the effect of the epidermal growth factor receptor (EGFR) small molecule tyrosine kinase inhibitor gefitinib on five JPA primary cell-cultures. Due to a lack of established cell-lines of JPA very few in vitro drug sensitivity assays have been performed. In this study we have succeeded in propagating short-term primary cell-cultures established from surgical specimens. The effect of gefitinib on proliferation in JPA derived primary cell-cultures was measured by a standard tritiated thymidine incorporation assay. The level of expression of EGFR, the intended target of gefitinib, was measured by immunohistochemistry, flow cytometry and RT-PCR. Gefitinib was shown to inhibit proliferation in all five JPA cell-cultures tested, with IC-50's between 1.6 and 9.6 microM. However, EGFR protein and mRNA expression was undetectable. Further studies with cetuximab, an EGFR-specific inhibitory monoclonal antibody, showed no effect on proliferation in JPA. Based on these preclinical data, gefitinib may be a suitable salvage chemotherapy drug to explore further in those patients with JPA who have recurred after primary chemotherapy. Of interest, it appears that the anti-tumor effect of gefitinib in JPA cell-cultures may be mediated through a pathway other than EGFR inhibition.

Autofluorescence characterisation of isolated whole crypts and primary cultured human epithelial cells from normal, hyperplastic, and adenomatous colonic mucosa

Background/Aims: In vivo autofluorescence endoscopic imaging and spectroscopy have been used to detect and differentiate benign (hyperplastic) and preneoplastic (adenomatous) colonic lesions. This fluorescence is composed of contributions from the...

Additional Cyclin D1 gene copies associated with chromosome 11 aberrations in cutaneous malignant melanoma

Cyclin D1 (CCND1, PRAD1, bcl-1) is associated with progression through G1 and entry into S phase of the cell cycle, as a partner of cyclin-dependent kinases (CDKs). The aim of this study was to evaluate cytogenetic aberrations of the gene locus at chromosome 11q13 in cutaneous malignant melanoma. Fluorescence in situ hybridisation (FISH) was applied on metaphase spreads of short-term primary cell cultures as well as on 5 microm paraffin tissue sections of primary melanomas and melanoma metastases, and on melanoma cell lines (C32, WM 266-4, HT 144, RPMI 7951, SK MEL 28). For FISH analysis DNA probes specific for Cyclin D1, centromere 11 and painting probes for whole chromosome 11 were used. FISH revealed additional Cyclin D1 gene copies in relation to centromere 11 copy numbers in 9/19 (47%) primary melanomas and in 7/20 (35%) melanoma metastases. The melanoma cell line SK MEL 28 also showed extra Cyclin D1 gene copies. A 24-colour FISH (mFISH) investigation revealed translocated chromosome 11 material to chromosomes 12, 14, 15, 20, 21, 22 and Y in these cases, respectively. Positivity for Cyclin D1 protein was detected by immunostaining in 17/19 (89%) primary melanomas (10 weak, 7 strong expression) and in 9/12 (75%) melanoma metastases (5 weak and 4 strong). All primary melanomas and 3/4 examined metastases with additional Cyclin D1 gene copies concomitantly revealed positive protein staining. Based on these results Cyclin D1 could well be involved in the pathogenesis of a subset of malignant melanoma. Additional studies will have to elucidate the role of further genes located at chromosome 11q13.

Effects of extracellular iron concentration on calcium absorption and relationship between Ca2+and cell apoptosis in Caco-2 cells

To determine the method of growing small intestinal epithelial cells in short-term primary culture and to investigate the effect of extracellular iron concentration ([Fe3+]) on calcium absorption and the relationship between the rising intracellular calcium concentration ([Ca2+]i) and cell apoptosis in human intestinal epithelial Caco-2 cells. Primary culture was used for growing small intestinal epithelial cells. [Ca2+]i was detected by a confocal laser scanning microscope. The changes in [Ca2+]i were represented by fluorescence intensity (FI). The apoptosis was evaluated by flow cytometry. Isolation of epithelial cells and preservation of its three-dimensional integrity were achieved using the digestion technique of a mixture of collagenase XI and dispase I. Purification of the epithelial cells was facilitated by using a simple differential sedimentation method. The results showed that proliferation of normal gut epithelium in vitro was initially dependent upon the maintenance of structural integrity of the tissue. If 0.25% trypsin was used for digestion, the cells were severely damaged and very difficult to stick to the Petri dish for growing. The Fe3+ chelating agent desferrioxamine (100, 200 and 300 micromol/L) increased the FI of Caco-2 cells from 27.50+/-13.18 (control, n=150) to 35.71+/-13.99 (n=150, P<0.01), 72.19+/-35.40 (n=150, P<0.01) and 211.34+/-29.03 (n=150, P<0.01) in a concentration-dependent manner. There was a significant decrease in the FI of Caco-2 cells treated by ferric ammonium citrate (FAC, a Fe3+ donor; 10, 50 and 100 micromol/L). The FI value of Caco-2 cells treated by FAC was 185.85+/-33.77 (n=150, P<0.01), 122.73+/-58.47 (n=150, P<0.01), and 53.29+/-19.82 (n=150, P<0.01), respectively, suggesting that calcium absorption was influenced by [Fe3+]. Calcium ionophore A23187 (0.1, 1.0 and 10 micromol/L) increased the FI of Caco-2 cells from 40.45+/-13.95 (control, n=150) to 45.19+/-21.95 (n=150, P<0.01), 89.87+/-43.29 (n=150, P<0.01) and 104.64+/-51.07 (n=150, P<0.01) in a concentration-dependent manner. The positive apoptotic cell number of the Caco-2 cells after being treated with A23187 increased from 0.32% to 0.69%, 0.90% and 1.10%, indicating that the increase in the positive apoptotic cell number was positively correlated with [Ca2+]i. Ca2+ absorbability is increased with the decrease of extracellular iron concentration Fe3+ and hindered with the increase of Fe3+ consistence out of them. Furthermore, increase of [Ca2+]i can induce apoptosis in Caco-2 cells.

Development and transfection of short-term primary cell cultures from Brugia malayi.

Development and transfection of short-term primary cell cultures from Brugia malayi.

Chromosome 7 aneusomy. A marker for metastatic melanoma? Expression of the epidermal growth factor receptor gene and chromosome 7 aneusomy in nevi, primary malignant melanomas and metastases.

Receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR) play an important role in a variety of malignant neoplasias, making the search for aberrations in the relevant chromosomes an important issue. Differential expression of the EGFR gene was investigated by reverse transcriptase (RT)-PCR on tissue samples of normal skin, nevi, primary melanomas, and melanoma metastases. The EGFR gene is located on chromosome 7p12.3-p12.1. To determine the number of chromosomes 7 in cell nuclei of the mentioned tissue samples we performed fluorescence in situ hybridization (FISH) on touch preparations, using a DNA probe that hybridizes specifically to the centromeric region of chromosome 7. Additionally, chromosome 7 number in interphase nuclei was determined in short-term primary cell cultures of nevi, primary melanomas, and metastases. The highest EGFR gene expression frequency was found in melanoma metastases. By FISH we detected the highest fraction of cell nuclei with more than two chromosomes 7 in the group of metastases. Our results suggest that overexpression of the EGFR gene might play an important role in metastasis of malignant melanoma. This is well reflected by polysomy 7, possibly accounting for an increased EGFR gene copy number.

Chromosome 7 Aneusomy. A Marker for Metastatic Melanoma?: Expression of the Epidermal Growth Factor Receptor Gene and Chromosome 7 Aneusomy in Nevi, Primary Malignant Melanomas and Metastases

Receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR) play an important role in a variety of malignant neoplasias, making the search for aberrations in the relevant chromosomes an important issue. Differential expression of the EGFR gene was investigated by reverse transcriptase (RT)-PCR on tissue samples of normal skin, nevi, primary melanomas, and melanoma metastases. The EGFR gene is located on chromosome 7p12.3-p12.1. To determine the number of chromosomes 7 in cell nuclei of the mentioned tissue samples we performed fluorescence in situ hybridization (FISH) on touch preparations, using a DNA probe that hybridizes specifically to the centromeric region of chromosome 7. Additionally, chromosome 7 number in interphase nuclei was determined in short-term primary cell cultures of nevi, primary melanomas, and metastases. The highest EGFR gene expression frequency was found in melanoma metastases. By FISH we detected the highest fraction of cell nuclei with more than two chromosomes 7 in the group of metastases. Our results suggest that overexpression of the EGFR gene might play an important role in metastasis of malignant melanoma. This is well reflected by polysomy 7, possibly accounting for an increased EGFRgene copy number.

Cytotoxicity assessment of antibiofouling compounds and by-products in marine bivalve cell cultures

Short-term primary cell cultures were derived from adult marine bivalve tissues: the heart of oyster Crassostrea gigas and the gill of clam Ruditapes decussatus. These cultures were used as experimental in vitro models to assess the acute cytotoxicity of an organic molluscicide, Mexel-432 ®, used in antibiofouling treatments in industrial cooling water systems. A microplate cell viability assay, based on the enzymatic reduction of tetrazolium dye (MTT) in living bivalve cells, was adapted to test the cytotoxicity of this compound: in both in vitro models, toxicity thresholds of Mexel-432 were compared to those determined in vivo with classic acute toxicity tests. The clam gill cell model was also used to assess the cytotoxicity of by-products of chlorination, a major strategy of biofouling control in the marine environment. The applications and limits of these new in vitro models for monitoring aquatic pollutants were discussed, in reference with the standardized Microtox ® test.

Inhibitory Effects of an Organic Molluscicide on Byssal Thread Development in Zebra Mussels

Short-term primary cell cultures were derived from adult marine bivalve tissues: the heart of oyster Crassostrea gigas and the gill of clam Ruditapes decussatus. These cultures were used as experimental in vitro models to assess the acute cytotoxicity of an organic molluscicide, Mexel-432®, used in antibiofouling treatments in industrial cooling water systems. A microplate cell viability assay, based on the enzymatic reduction of tetrazolium dye (MTT) in living bivalve cells, was adapted to test the cytotoxicity of this compound: in both in vitro models, toxicity thresholds of Mexel-432 were compared to those determined in vivo with classic acute toxicity tests. The clam gill cell model was also used to assess the cytotoxicity of by-products of chlorination, a major strategy of biofouling control in the marine environment. The applications and limits of these new in vitro models for monitoring aquatic pollutants were discussed, in reference with the standardized Microtox® test.

Endothelin-1 mRNA in glomerular and epithelial cells of kidney.

To examine the question of the tubular localization of renal endothelin-1 (ET-1) mRNA, cDNA generated by reverse transcription of isolated rat tubule RNA was amplified by polymerase chain reaction using rat ET-1-specific oligonucleotides. Product identity was determined by restriction enzyme digestion or direct product sequencing. ET-1 mRNA was found to increase in renal tissue in a corticomedullary direction. High levels of ET-1 mRNA were found in dissected glomeruli and in juxtaglomerular cells in short-term primary culture. Among tubule segments, ET-1 mRNA was most abundant in inner medullary collecting ducts (IMCD), but products were also found with cDNA derived from proximal convoluted and straight tubules, thick ascending limbs, and outer medullary collecting ducts. In kidneys of untreated, homozygous Brattleboro rats, the increase of ET-1 mRNA along the corticomedullary axis as well as the preponderance of tubular ET-1 mRNA in IMCD was not observed. Our data show that ET-1 mRNA is present in all nephron segments studied and that its expression may be dependent on the functional state of the kidney. Our results are consistent with the proposal that ET-1 modifies tubular function in an autocrine or paracrine fashion.

Pharmacological block of Ca(2+)-activated Cl- current in rat vascular smooth muscle cells in short-term primary culture.

Ca(2+)-activated Cl- currents were studied in isolated cells from rat portal vein smooth muscle in short-term primary culture using the whole-cell patch-clamp technique. Cl- currents can be activated separately by Ca2+ release from intracellular stores (in response to external applications of caffeine or noradrenaline) and by Ca2+ influx through voltage-dependent Ca2+ channels. The effects of several Cl- channel blockers and of spironolactone (a substance known to reduce internal Ca2+ loading) on both Cl- and Ca2+ currents were examined. Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), anthracene-9-carboxylic acid (9-AC) and diphenylamine-2,2'-dicarboxylic acid (DPC) inhibited the Ca(2+)-activated Cl- current (IC50 values between 16.5 and 306 microM) with no effects on the inward Ca2+ current and on internal Ca2+ loading (testing by measuring the Ca(2+)-activated K+ current). These results indicate that the inhibition of Cl- current by these compounds is due to a direct interaction with the Cl- channel. In contrast, spironolactone inhibited both K+ and Cl- currents (IC50 = 7.6 microM) by reducing the amount of Ca2+ located in the internal stores, whereas the Cl- current activated by Ca2+ current through T-type Ca2+ channels was unchanged. This preparation and the protocols developed in this study appears to be appropriate for analysis of substances interfering with Cl- channels or intracellular Ca2+ stores.

Serum Factors Induce C-fos Expression and Rapid Cell Proliferation in Adolescent But Not in Infant Rat Proximal Tubule Cells

Kidney epithelial cells in short-term primary culture have been studied with regard to proliferative rate and expression on the c-fos protooncogene. The experiments were performed on subconfluent renal proximal tubule cells isolated from infant and adolescent rats. Proliferation was determined by 3H-thymidine autoradiography and nuclear content of c-fos protein by semiquantitative immunofluorescence. The basal proliferative rates in infant and adolescent renal proximal tubule cells were the same after 48 h of primary culture in Dulbecco's modified Eagle's medium with 10% FCS. Serum deprivation for 24 h caused a significant growth inhibition in both infant and adolescent cells. C-fos was expressed to the same extent in infant and adolescent serum-deprived cells. The rapid response to the addition of serum was markedly different in infant and adolescent cells. In adolescent cells, addition of serum led to a transient significant increase in the nuclear expression of c-fos protein, reaching a peak at 60 min. No increase in c-fos was seen in infant cells. In adolescent cells, the rate of proliferation increased 11-fold and 3H-thymidine labeling index reached 26.7 +/- 4.3%. In infant cells, the proliferative response to serum addition was significantly lower; the labeling index reached only 4.2 +/- 1.2%. It could be excluded that the attenuated response in infant cells was due to cell death or impaired metabolic function. The results imply that the principles of growth regulation change postnatally.

Effect of cell purity, cell concentration, and incubation conditions on rat testis leydig cell steroidogenesis

This study examines the effects of cell purity and incubation conditions on testosterone production by rat testis Leydig cells in short-term primary culture. Both basal and luteinizing hormone (LH)-stimulated testosterone production were affected by the purity of the cell preparation, i.e., as the purity of the cell preparation was increased the amount of testosterone produced per Leydig cell was also found to increase. The stimulation ratio of testosterone production, calculated as the secretion of testosterone in the presence of LH (100 ng/ml) divided by the basal secretion of testosterone, increased with the increase in plating density (20,000 to 200,000 cells per well). This pattern of change was independent of the vessel and volume of incubation. In terms of the absolute amount of testosterone produced, increasing the plating density led to a decrease in the amount of steroid produced both basally and in response to LH. Composition of the incubation medium also had an effect on testosterone production; phenol red and sodium bicarbonate exerted negative effects. At all temperatures studied (4 degrees, 24 degrees, 34 degrees, and 37 degrees C), LH increased testosterone production and the degree of stimulation increased with temperature. We conclude that cell purity and incubation conditions markedly affect rat Leydig cell steroidogenesis in vitro. Furthermore, the manner in which the results are presented can affect their interpretation.

On the active form of 4‐aminopyridine: block of K+ currents in rabbit Schwann cells.

1. The blocking action of 4-aminopyridine (4-AP) on the outward potassium currents evoked by depolarization of rabbit Schwann cells in short-term primary culture was studied with the whole-cell configuration of the patch-clamp method. 2. We have determined the apparent equilibrium dissociation constant, K, for the action of 4-AP to block potassium currents at a series of different extracellular and intracellular pH values. 4-Aminopyridine is an organic base and exists in both charged and uncharged forms in aqueous solution. Changes in the pH of the extracellular and intracellular solutions therefore also change the extracellular and intracellular proportions of these two forms, and the values of K that were obtained were found to depend in a consistent way on both the extracellular and the intracellular pH. 3. At alkaline extracellular pH, K was decreased. At acidic extracellular pH, K was increased. In contrast, increasing the intracellular pH from 7.2 to 8.1 reduced the apparent potency of extracellularly applied 4-AP (i.e. increased K), and decreasing the intracellular pH (to 6.4) increased this apparent potency (i.e. decreased K). 4. The 4-AP analogues, 2-aminopyridine, 3-aminopyridine and 3,4-diaminopyridine, were also tested. At half-block of the potassium current, the intracellular concentration of the cationic form of the various aminopyridines (applied extracellularly at pH 7.2) varied by a factor of less than five, whereas that of the uncharged form varied by a factor of over 700. 5. The results are inconsistent with the hypothesis that the cationic form of the aminopyridines, acting from the extracellular solution, contributes in any substantial way to potassium channel block. It also seems unlikely that the uncharged form, acting either extracellularly or intracellularly, is solely responsible for the block. However, the results as a whole are consistent with the idea that it is the cation acting from the intracellular side that blocks the 4-AP-sensitive potassium channel and that the affinity with which 4-AP blocks the channel depends on the intracellular pH. The results would be explained if the cation competes with protons for a binding site that has an apparent pKa of about 7.0. 6. The results, nevertheless, are not inconsistent with the possibility that both the uncharged form and the intracellular charged form of 4-AP are active in blocking the 4-AP-sensitive potassium channel.(ABSTRACT TRUNCATED AT 400 WORDS)