Isolation and culture of the terminally differentiated adult mammalian ventricular cardiac muscle cell

Summary We have carried out systematic studies to optimize and standardize methodology to isolate and culture the adult rat ventricular cardiac muscle cell. Four hearts were perfused simultaneously with a calcium-free medium containing collagenase. The ventricular tissue was then minced and further digested to liberate individual cells. Approximately 16 million rod-shaped muscle cells were obtained. The plating efficiency has been greatly improved by culturing the cells in a conditioned medium prepared from a rabbit corneal cell line. This medium also contained added fetal bovine serum, essential and nonessential amino acids, vitamins, insulin, transferrin, and 25 trace minerals. The culture flasks were precoated with rat-tail collagen. Fibroblast contamination was virtually eliminated by including cytosine arabinoside in the medium during the first 7 d of culture. After this time the cells could be cultured in the absence of serum in a chemically defined medium composed of MEM, vitamins, nonessential amino acids, and trace minerals. They continued to contract spontaneously and do well in this medium for at least 3 d thereafter. This improved methodology resulted in a reproducible culture system with improved plating efficiency. It provided a new and unique system to study the structure and function of the adult mammalian ventricular cardiac muscle cell. This investigation was supported by Grant HL 25873 from the National Institutes of Health, Bethesda, MD.     

Isolation and long-term culture of rat,rabbit, and human nasal turbinate epithelial cells

Summary Nasal turbinate epithelial cells were isolated from rats, rabbits, and humans using either a surgical or an in situ enzyme incubation technique. The culture conditions that permit optimal cell attachment and selective growth of the nasal epithelial cells were determined. These conditions will permit the long-term culture of these cells where typically 20 to 30 population doublings were observed. Differences between rat and human nasal epithelial cells were seen in substrate requirements, colony-forming efficiency, and response to fetal bovine serum and bovine serum albumin. These methodology and results will permit mechanistic studies of normal and abnormal cellular function and comparative response studies between nasal epithelial cells from rats and humans. This work was supported under U.S. Environmental Protection Agency contract 68-02-4032.     

Isolation and characterization of rabbit ovarian surface epithelium,granulosa cells,and peritoneal mesothelium in primary culture

Summary Mammalian ovarian surface epithelial (OSE) cells and peritoneal mesothelial (PM) cells have a common embryologic origin, yet certain morphologic and histochemical characteristics are different in the adult. In this study, a two-step culture method was developed to examine the characteristics of these two cell types in vitro. OSE, PM, and ovarian granulosa (GC) cells were isolated from estrous rabbits and cultured for 6 d in 5% serum-supplementedd-valine medium (to inhibit fibroblast growth), then incubated for a further 2 d in serum-free McCoy's 5A medium. This study showed that rabbit OSE and PM cells in vitro maintained certain in vivo morphologic characteristics; OSE cells exhibited distinct cell borders and abundant microvilli of homogeneous size and shape, whereas PM cells were characterized by obscure cell borders and abundant microvilli of heterogeneous form. GC in vitro exhibited overlapping cell borders and sparse microvilli of homogeneous structure. This study showed for the first time that cultured rabbit OSE and PM cells, but not GC, contain distinct filaments of cytokeratin 18. In addition, rabbit OSE cells and GC, but not PM cells, contained 17β-hydroxysteroid dehydrogenase. However, only GC contained delta 5-3β hydroxysteroid dehydrogenase. OSE, PM, and GC maintained their ultrastructural and histochemical characteristics in serum-free medium. These results suggest that rabbit OSE cells in vitro could be distinguished from PM cells by histochemical and ultrastructural differences. Furthermore, because these characteristics were not altered in serum-free medium, the two-step culture method will be valuable in further hormonal studies of these cells in vitro. This work was supported in part by Grant No. 202-3192 from the University of South Dakota Parsons Fund     

Culture and characterization of fetal human atrial and ventricular cardiac muscle cells

Summary Atrial and ventricular cardiac muscle cells isolated from 14- to 18-wk old fetal human hearts were grown in culture and characterized. Once established in culture the flattened cells contracted spontaneously and possessed differentiated ultrastructural characteristics including organized sarcomeres, intercalated discs, and transverse tubules with couplings. Atrial granules were present in the cultured atrial cells. Some cultured ventricular myocytes also contained electron-dense granules associated with Golgi cisternae, which were similar in size and appearance to atrial granules. The cultured ventricular myocytes divided and expressed the genes for thymidine kinase, histone H4, myosin heavy chain, muscle-specific creatine kinase, atrial natriuretic factor, and insulin-like growth factor II. These results establish that differentiated fetal human heart muscle cells can be cultured in sufficient quantities for biochemical, molecular, and morphological analyses. This work was supported by a postdoctoral fellowship from the American Heart Association, Louisiana Affiliate (JBD) and the National Institutes of Health, Bethesda, MD (HL-35632) (WCC).     

DNA synthesis of adult mammalian cardiac muscle cells in long-term culture

Adult rat cardiac ventricular muscle cells were isolated and cultured in monolayer for 30-45 days. Most of the cardiac muscle cells undergo external and internal structural alterations, resembling embryonic/neonatal cardiac muscle cells in culture (Nag and Cheng, 1981; Nag et al., 1983). These cultured cells underwent DNA synthesis and mitosis as revealed by autoradiography studies that involved the exposure of the cells to [3H]-thymidine for 24 hr prior to the termination of the culture at selected intervals. During the first week of culture, cardiac muscle cells showed less than 5% labeled cells. The labeling index of myocytes attained a peak in the second week of culture, exhibiting approximately 23% labeled cells. The labeling indices of cardiac muscle cells declined over the period of 30 days of culture. During the end of the incubation period, approximately 4% of the myocytes were labeled. When the extent of the total cell population involved in DNA synthesis was examined by exposing the cells to [3H]-thymidine continuously for long periods of time, it was observed that approximately 26% of the cardiac muscle cells regained the capacity for DNA synthesis during 1-10 days of culture. From day 1 to day 14, approximately 29% of the total muscle cell population was labeled. When the cells were exposed to the radioactive isotope continuously for 30 days, approximately 31% of the cells incorporated radioactive isotope, showing their capacity for DNA synthesis. Approximately 90% of the cardiac muscle cells in long-term culture contained more than one nucleus. The nuclei were often observed in multiples of two. Labeled mitotic apparatus was observed in cardiac myocytes, indicating the replication of DNA, followed by karyokinesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

小鼠骨片间充质干细胞分离培养及鉴定

目的建立小鼠骨片间充质干细胞（MSC）分离培养及扩增的方法。方法取小鼠胫骨和股骨,洗去骨髓后,用胶原酶I消化疏松骨密质,利用MSC具有迁徙和贴壁生长的能力进行分离。并对获取的细胞进行流式鉴定和诱导分化。结果培养2d小鼠骨片边缘爬出成纤维样细胞,呈克隆和鱼群样生长,并可以进行持续传代培养。流式鉴定结果显示这群细胞表达MSC标志Scall（92．7％）,CD29（98．4％）,CD90（91．6％）,不表达造血细胞标志CD34（1．57％）,CD45（3．99％）,CD11b（0．63％）,并可成功诱导分化成骨细胞和脂肪细胞。结论成功建立从小鼠骨片中获得MSC的方法,为实验研究提供可靠的细晌实源．     

Isolation,characterization, and in vitro culture of larval and adult epidermal cells of the frogXenopus laevis

Summary Methods for the isolation and in vitro culture of larval and adultXenopus laevis epidermal cells have been developed. Epidermal cells of stage 52–54 tadpoles and adult epidermal cells were enzymatically dissociated and purified (98%) by Percoll-density centrifugation and unit-gravity sedimentation. Both cell types attached on fibronectin-coated dishes and proliferated for 1 wk when the proper medium was used. There were four significant differences between larval and adult cells: a) Adult cells had a greater buoyant density than larval cells. b) Keratin synthesis patterns were markedly different. c) A combination of medium F12 and Eagle's minimum essential medium was optimal for growth of larval cells whereas MCDB151 medium was optimal for adult cells. d) Adult cells needed fetal bovine serum (>5%) whereas larval cells grew without fetal bovine serum. In contrast to these differences, larval and adult cells had two similar properties: a) Insulin had a potent effect on the growth of both cells, and b) The optimal Ca⁺⁺ concentration for cell growth was quite low for both cell types; 0,1 mM for larval cells and below 0.05 mM for adult cells. These results suggest that low Ca⁺⁺ levels are essential for both cornifying (adult) and uncornifying (larval) amphibian keratinocytes. The culture techniques described herein for larval and adult epidermal cells provide a new in vitro model for analyzing development of the epidermis during amphibian metamorphosis. This study was supported by grant (HD 24438) from the National Institutes of Health, Bethesda, MD.     

Isolation and culture of smooth muscle cells from human umbilical cord arteries

Summary A short method is described for obtaining a large number of pure vascular smooth muscle cells in culture. The smooth muscle cells were isolated from human umbilical cord arteries digested twice by an enzyme mixture of collagenase, trypsin, elastase, and DNAase with addition of α-tosyl-lysyl chloromethane. Primary cell culture and first subculture were not contaminated by endothelial cells, no Factor VIII being produced. The cultures consisted of smooth muscle cells as appeared from phase contrast and electron microscopy. Part of this study was supported by a scholarship from the Dutch Ministry of Education and Science and by the Leyden University Foundation.     

Isolation and culture of vascular smooth muscle cells from rat placenta

We developed a new separation method for isolating placental vascular smooth muscle cells (PVSMCs) from a rat in this study. Our method used the magnetic force between a magnet and ferrous ferric oxide (Fe₃O ₄) to make the separation and extraction processes easier and more efficient. From the first to sixth generation, the cells isolated using this protocol were identified as smooth muscle cells (SMCs) by their immunoreactivity to the SMC markers and by the “hill and valley” morphology. PVSMCs were exposed to angiotensin II (1 μmol/L) and resulted in sharply increased intracellular Ca ²⁺ concentration. Furthermore, activation of protein kinase C (PKC) increased concomitantly with a decrease in calponin expression. These results indicate that the isolated cells had biological activity. Our method of isolating PVSMCs from rat leads to isolation of cultured cells with activity and high purity. The approach will be useful in research studies on placental vascular diseases.     

Growth and ultrastructural characteristics of Citrus cells grown in medium containing NaCl

Changes in growth and structural properties of Citrus cell line Carvalhal acclimated to 100 mM NaCl in the medium were compared to unacclimated control cells and cells exposed to 100 mM NaCl. Transmission electron microscopy (TEM) showed presence of ring-shaped mitochondria, increase in the number of amyloplasts and lipid bodies, higher cell wall thickness and partitioned vacuoles in acclimated cells.     

TPA has no influence on the expression of myosin heavy chain isoforms in cultured adult cardiac muscle cells.

The effect of a tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), on the expression of myosin heavy chain isoforms in cultured rat cardiac ventricular muscle cells was studied. The previous preliminary report [Claycomb WC (1988): "Biology of Isolated Adult Cardiac Myocytes." In Clark WA, Decker RS, Borg TK (eds): New York: Elsevier, pp 284-287] indicated that TPA turns off the expression of myosin heavy chain genes in cultured adult cardiac myocytes. Electrophoretic and immunocytochemical analyses were carried out in the present studies. The myosin heavy chain isoform profiles of cardiac myocytes exposed to TPA at concentrations of 50-250 ng/ml culture medium for varying periods were similar to those of controls that were grown in the absence of TPA, showing predominant isoform V1. Immunofluorescence microscopy with monoclonal antibodies to cardiac ventricular isomyosin revealed the structural organization of myosin in TPA-treated cells. The organization of myosin was variable among different myocytes and within a single myocyte. Immunofluorescence microscopy was extended to the examination of the organization of alpha-actinin which did not differ from that of myosin in some myocytes. In contrast to the previous report [Claycomb, 1988], this study has demonstrated that TPA has no influence on the expression of myosin heavy chain isoforms in cultured adult ventricular cardiac muscle cells.     

Isolation and long-term culture of gallbladder epithelial cells from wild-type and CF mice

Summary Mice with targeted disruption of the cftr gene show pathophysiologic changes in the gallbladder, which correlate with hepatobiliary disease seen in cystic fibrosis patients. As gallbladder epithelium secretes mucin, and as this epithelium consists of a relatively homogenous cell type, study of CFTR function in these cells would be beneficial to delineate the complex cellular functions of this protein. The size and anatomic location of the murine gallbladder makes such studies difficult in vivo. Therefore, the need exists for in vitro models of gallbladder epithelium. We describe a method to isolate and culture murine gallbladder epithelium from wild-type and CF mice. Cells were grown in a monolayer on porous inserts over a feeder layer of fibroblasts. These nontransformed cells can be successively passaged and maintain a well-differentiated epithelial cell phenotype as shown by morphologic criteria, characterized by polarized columnar epithelial cells with prominent microvilli and intercellular junctions. Organotypic cultures showed columnar cells simulating in vivo morphology. This culture system should be valuable in delineating cellular processes relating to CFTR in gallbladder epithelium.     

Morphological and ultrastructural characterization of sea urchin immune cells

The free circulating coelomocytes in the coelomic cavity of echinoderms are considered to be immune effectors by phagocytosis, encapsulation, cytotoxicity, and by the production of antimicrobial agents. Although echinoderms (especially sea urchin embryo) have been used as a model organisms in biology, no uniform criteria exist for classification of coelomocytes in echinoderms, and few studies have reported about the biological functions of their coelomocytes. Hence, we study the coelomocytes in the echinoid sea urchin, Paracentrotus lividus, and describe their morphological and ultrastructural features using light and transmission electron microscopes. We classify the coelomocytes of P. lividus into red spherule and colorless spherule cells, small cells, vibratile cells, and phagocytic cells; petaloid and filopodial cells. To our knowledge, this is the first report describing ultrastructural details of the coelomocytes of P. lividus. J. Morphol. 276:583–588, 2015. © 2015 Wiley Periodicals, Inc.     

Trout sertoli and leydig cells: Isolation,separation, and culture

Trout testes at various stages of maturation were dissociated by perfusion at 12°C with collagenase plus pronase and then with collagenase alone, followed by slight shaking overnight in 1% bovine albumin. This step provided a suspension of isolated somatic and germ cells, clusters of interstitial cells, and either intact spermatogenetic cysts (meiotic testes) or clusters of Sertoli cells (other testes). Most of the spermatozoa were removed from the testis cell suspension by centrifugation in Percoll (density 1.065 g/ml). Sertoli and Leydig cells were prepared by a two-step separation method: (1) the testis cell suspension was separated by sedimentation at unit gravity into “isolated cell” and “cell cluster” populations; (2) these populations were fractionated by isopyknic centrifugation in Percoll gradients. In terms of somatic cell composition, a nearly pure Sertoli cell (clusters) population was obtained between 1.017 and 1.033 g/ml and a Leydig cell (clusters) enriched population of between 1.033 and 1.048 g/ml (testes resuming spermatogenesis) or 1.048 and 1.062 g/ml (other testes). These various cell populations were cultured in modified Leibovitz L15 medium for 10–15 days. When seeded, the Sertoli cells had a normal ultrastructure that remained unchanged for at least 10 days, and the steroidogenic activity of Leydig cells could be stimulated by salmon gonadotropin. Leydig cells remained 3β-HSD positive and produced progesterone and 17α, 20β-OH progesterone for at least 11 days. This study points out that viable and differentiated trout somatic testicular cells can be prepared and cultured for several days.     

Isolation,characterization, and long-term culture of fetal bovine tracheal epithelial cells

Summary Epithelial cells were isolated from fetal bovine trachea by exposing and stripping the mucosal epithelium from the adjacent connective tissue. The tissue was minced and enzymically dissociated in Ca-Mg-free medium containing dispase and dithiothreitol. The stripping procedure and selective trypsinization produced epithelial cell cultures free of fibroblasts. Seeded on plastic, the plating efficiency was 21.5% with a doubling time of 24 h. Dome formation, evidence of occluding junctions and active ion transport characteristic of epithelial cells, was common. Growth of the cells on glass, collagen, and Engelbreth-Holm-Swarm (EHS) substrate demonstrated a striking difference in morphology. Cells grown on EHS presented a more distinctly three-dimensional growth pattern and many more microvilli when compared to cells grown on glass or collagen. The cells retained their epithelioid characteristics through more than 30 passages as shown by the presence of distinct apical and basolateral membranes, tight junctions, and positive keratin staining. This study was supported in part by grants BRSG S07 RR05408-25, Biomedical Research Support Grant Program, Division of Research Resources, by ES 00159, Center Grant, National Institutes of Environmental Health Sciences, by R23-HL37621, New Investigator Award, National Heart, Lung and Blood Institutes, National Institutes of Health, and by the Health Effects Institute, an organization jointly funded by the U.S. Environmental Protection Agency (Assistance Agreement X-8120059) and automotive manufactures. The contents do not necessarily reflect the views of policies of HEI, EPA, or automotive manufacturers.     

Establishment and morphologic characterization of normal human endometrium in vitro

Summary Tissue culture offers a model system with which to study the endocrine-mediated growth, differentiation, and metabolic activities of the endometrium. We have established and continue to maintain monolayer cultures of normal human endometrial epithelial cells from each phase of the menstrual cycle. At present, eight proliferative, two secretory, and two menstrual phase cultures have been established. These have been passed at least three times. One proliferative phase culture has been growing for 18 mo, and passed 10 times. Colonies of epithelioid cells as well as single cells appear in the cultures within 2 to 8 h of initial culture and maintain this appearance throughout long-term growth. The cells are periodic acid Schiff positive for carbohydrates and positive for keratin, an immunochemical marker for epithelial tissues. Studies comparing the ultrastructure of the cultures with fresh endometrial tissue revealed morphologic features common to both, including prominent nucleoli, Golgi, mitochondria-rough endoplasmic reticulum complexes, and abundant glycogen. The cells are not tumorigenic in the nude mouse and do not form colonies on soft agarose, confirming the nonneoplastic identity of the cells. The opinions and assertions contained herein are those of the authors and are not to be construed as official or representing those of the Uniformed Services University of the Health Sciences, The Department of the Navy, or the Department of Defense. This project was supported by the following grants: C08509 from the Uniformed Services University and Clinical Investigation Study Protocol 82-06-1804, Naval Hospital, Bethesda. Presented at the Twenty-Second Annual Meeting of the Armed Forces District of the American College of Obstetricians and Gynecologists, 9–13 October 1983; Founders Award for the Best Paper on a Basic Scientific Subject.     

Isolation of adult canine venous endothelium for tissue culture

Summary In order to provide autologous adult endothelial cells for the production of cell-lined artificial vascular prostheses, we have developed a method for harvesting large numbers of cells with minimal contamination by other cellular types. In this technique, the vein to be stripped is isolated, removed, and everted over a stainless steel rod. After washing, the vein is incubated in trypsin-EDTA solution followed by collagenase and the endothelial cells flushed off with a stream of culture medium. With care and appropriate timing, the endothelium can be selectively removed leaving the underlying basal lamina intact. This research was supported by National Heart, Lung and Blood Institute Contract NIH-NO1-HV-2054 and Grant HL23345.     

Human microvessel endothelial cells: Isolation,culture and characterization

Summary Over recent years, interest in endothelial cell biology has increased dramatically with our ability to grow and study endothelial cellsin vitro. While large veins and arteries remain a quick and convenient source of endothelial cells, the great morphological, biochemical and functional heterogeneity that endothelial cells express has necessitated the development of techniques to isolate microvessel endothelial cells from different tissues to create more realisticin vitro models. The majority of isolation procedures employ selective methods to enrich microvessel endothelial cells from tissue homogenates directly, or after a period in culture. These include sieving/filtration, manual weeding, isopycnic centrifugation, selective growth media, and the use of flow cytometry or magnetic beads coupled with specific endothelial cell markers. The establishment of pure endothelial cell populations is important for studying their biochemistry and physiology and there are many morphological, immunological and biochemical criteria which can be used to characterize human endothelial cells. These range from classical markers such as von Willebrand Factor and angiotensin-converting enzyme to novel markers like platelet endothelial cell adhesion molecule-1 (CD31) and the expression of E-selectin on cytokine-activated endothelial cells.