Isolation,biochemical characterization,and culture of lung type II cells of the rat

A method is described for the isolation of rat lung epithelial Type II cells using trypsin digestion of tissue to release cells for subsequent separation by Percoll gradient centrifugation. Both the concentration of trypsin and the age (body weight) of the rat affect the yield from primary digestion and the final number of Type II cells obtained. A lung weighing 1 g from a 200 g rat yields approximately 30 × 10⁶ washed Type II cells (approximately 25% of the total estimated lung population). These cells have a plating efficiency of 40–50% after 48 h of culture. The cells have a high alkaline to acid phosphatase ratio (usually >4.0) compared with that of alveolar macrophages (0.1) and accumulate putrescine by an active transport mechanism with an apparent K_M between 8 and 14 μM. Together with studies of [³H]thymidine uptake into DNA, which is maximal between 48 and 72 h of culture, these quantitative measurements form a good basis for investigating the interactions between a number of chemical agents and Type II cells in vitro.     

Type II alveolar epithelial cells in suspension: separation by density and velocity

A method has been developed for obtaining a satisfactory yield of high purity Type II alveolar epithelial cell suspensions from rat lungs that does not require adherence to a surface. The technique involves buoyant density gradient sedimentation followed by unit gravity velocity sedimentation, thereby achieving separation by density and by size, respectively. Isolated, lavaged and perfused rat lungs are inflated first with albumin-fluorocarbon emulsion and then with elastase solution. The lungs are minced and sequentially filtered. The resultant crude cell mixture (48% Type II cells) is layered onto a discontinuous metrizamide gradient and centrifuged. The broad Type II cell band (67% Type II cells) is loaded onto a unit gravity cell separator and allowed to settle for 2.5 h. We obtained pooled purified fractions containing about 9×10⁶ cells/rat which are >90% Type II cells and >95% viable by trypan blue dye exclusion. These Type II cells form confluent monolayers and exhibit active transport within three days when plated in primary culture. This preparative method (which avoids adherence of cells to a surface and subsequent re-exposure to proteolytic enzymes) results in high purity, viable Type II cell suspensions with a yield sufficient for most experimental applications.     

新生大鼠心肌细胞的原代培养及鉴定

目的：探讨新生大鼠心肌细胞原代培养的方法,培养存活率高和纯度高的心肌细胞。方法：无菌条件下取出出生1~2 d的SD大鼠心脏,用2.5 g/L胰蛋白酶和 2.0 g/L Ⅱ型胶原酶等量混合液在37℃条件下分次消化心肌组织。以差速贴壁并将时间延长至90 min纯化心肌细胞,48 h后换液。结果： 将心肌细胞24 h贴壁生长,于2~3 d心肌细胞伸出伪足,形成细胞簇,同步搏动,频率约80~130次/min,存活率为（93.9±2.8）%,纯度为（91.9±2.2）%。结论：以本实验的方法培养的心肌细胞存活率高、纯度高,是一种较为理想的原代心肌细胞培养方法。     

Isolation and properties of type II alveolar cells from rat lung.

Type II alveolar cells can be isolated and partially purified from adult rat lung by a series of steps that includes enzymatic digestion of the lung with trypsin and separation of cells on a discontinuous albumin density gradient. The yield of the isolated type II cells depends on the supplier and the housing of the rats used to prepare the cells. With specific pathogen-free rats housed in a laminar flow hood, the yield was 20.3 x 10(6) cells per rat, of which 50 per cent were type II cells. With rats from 2 other suppliers and no special housing, the yields were 8.8 and 8.3 x 10(6) cells per rat, of which 67 and 65 per cent were type II cells. The ultrastructural appearance of the isolated cells was similar to that of cells from intact lung, except for some dilatation of the endoplasmic reticulum and the perinuclear space. Most cells (92 +/- 5 per cent) excluded the vital dye, trypan blue. The cells consumed O2 at the rate of 76 +/- 12 nmole per 10(6) cells per hour and released only 5.7 +/- 2.0 per cent of their lactate dehydrogenase, a cytoplasmic enzyme, into the medium after 1 hour of incubation. The isolated type II cells contained disaturated phosphatidylcholine, a major component of purified surface-active material. The cells, however, had a low glucose utilization compared to their O2 consumption, which may indicate an abnormality in the metabolism of glucose. This population of cells could be further purified to 89 per cent type II cells by unit gravity velocity sedimentation.     

Reproducible isolation of type II pneumocytes from fetal and adult rat lung using nycodenz density gradients.

Isolating fresh, relatively pure type II pneumocytes from the lung, particularly of fetal origin, is a difficult process. Separation by buoyant density gradient centrifugation has been used successfully to isolate adult type II cells. There is concern, however, that Percoll, a gradient medium that is commonly used for type II cell isolation, may be toxic to cells. We evaluated a new gradient medium, Nycodenz, that is (1) a true solution, (2) transparent, (3) not metabolized by cells, and (4) nontoxic to cells. Type II pneumocytes were isolated from 19- and 21-day gestation fetal and adult rat lung by elastase digestion and separated on preformed isotonic Nycodenz gradients (2 mL each of 27.6, 20.7, 13.8, and 4.6 (w/v) solutions). Type II pneumocytes were recovered from the density range 1.057-1.061 and identified by binding of FITC-conjugated and gold-complexed Maclura pomifera lectin. Cells derived from 19-day fetal lung contained abundant glycogen and reacted with a monoclonal antibody to the cytokeratins 8 and 18, which are markers of the fetal type II cell. Adult type II cells reacted with antibodies to cytokeratins 8, 18, and 19. Type II cell purity was 79.7 +/- 2.4%, 83.8 +/- 2.8%, and 82.6 +/- 1.8% (means +/- SEM) for 19- and 21-day gestation fetal and adult lung preparations, respectively. Cell viability was greater than 95%. The final cell yield for adult preparations was 17.8 +/- 2.7 x 10(6)/rat (means +/- SEM). To determine if the freshly isolated type II pneumocytes were functionally active, the incorporation of [3H]choline into phosphatidylcholine was measured. The percent saturation of phosphatidylcholine was high for both populations of freshly isolated cells. However, adult type II pneumocytes incorporated [3H]choline into phosphatidylcholine more rapidly than 21-day gestation fetal cells (5.97 x 10(-3) dpm/10(6) cells/h vs. 0.32 x 10(-3) dpm/10(6) cells/h, P less than .005). We have demonstrated that, using the Nycodenz isolation method, it is possible to obtain a high yield of relatively pure viable type II cells from fetal and adult lung that can be used for immediate study or cultured with an excellent plating efficiency (39 +/- 6.3%).     

Characterization of rat alveolar type II cells in vitro by immunological, biochemical, and morphological criteria

Using an anti-rat surfactant apoprotein antiserum which specifically reacts with cytoplasmic structures in alveolar type II cells on histopathology sections of rat lung, we have examined the immunoreactivity of pulmonary type II cells in vitro. Single cell suspensions of lung tissue were prepared from male Fischer 344 rats by intratracheal elastase digestion according to standard published methods. Cytocentrifuged preparations of the resulting cell suspensions revealed that approximately 40% of the cells stained positive for surfactant apoprotein using an immunoperoxidase staining technique. Without further cell fractionation steps, the cell suspensions were plated at colonial densities in growth medium. The cells that attached after 24 hours of incubation and at daily intervals were analyzed for surfactant apoprotein immunoreactivity as well as for proliferation, morphology, and phospholipid biosynthesis. The percentage of immunopositive cells increased with time from 75% at day 1 to 94% at 4 days after plating. This increase was paralleled by a linear increase in the number of immunopositive cells, which expanded into cell colonies. During the initial 5 days in vitro, the immunopositive cells retained their epithelial morphology and contained cytoplasmic osmiophilic bodies. Phospholipid biosynthesis by the isolated lung cells was analyzed and the data revealed that the rate of incorporation of 14C-choline into phosphatidylcholine increased with time in culture. These studies indicated that the anti-rat surfactant apoprotein antisera can be used to identify and quantitate functional alveolar type II cells in vitro. Thus the specific antisera may facilitate studies of type II cells undergoing various environmental alterations both in vivo and in vitro.     

Alkaline phosphatase: a marker of alveolar type II cell differentiation

In an effort to identify type II cells by a method independent of staining phospholipid inclusions, we evaluated a histochemical technique for alkaline phosphatase activity in normal rat lung, in freshly isolated type II cells, and in primary culture of type II cells. In the adult rat alveolus, alkaline phosphatase staining selectively identified type II cells, although nonciliated bronchiolar (Clara) cells and loose perivascular connective tissue also stained for alkaline phosphatase activity. In cell suspensions of type II cells and other dissociated lung cells, alkaline phosphatase staining correlated closely with the modified Papanicolaou technique and was particularly useful in distinguishing type II cells from alveolar macrophages. To determine if alkaline phosphatase was related to the differentiated phenotype of type II cells, we studied conditions known to affect other type II cell functions. When type II cells were cultured on plastic substrata, the intensity of alkaline phosphatase staining decreased with increasing time in culture. To quantitate the apparent decrease in alkaline phosphatase activity, we used a biochemical assay to study the expression of alkaline phosphatase by type II cells. The specific activity of alkaline phosphatase in type II cells declined with increasing time in tissue culture on plastic substrata. Alkaline phosphatase activity was maintained, however, by culturing cells on Englebreth-Holm-Swarm (EHS) tumor matrix. Cells that had reduced levels of alkaline phosphatase activity following 48 h of culture on plastic substrata could be "rescued" by removing them from the plastic substratum and reculturing them for 48 h on EHS matrix. Alkaline phosphatase activity was also increased by culturing type II cells in the presence of cAMP or sodium butyrate.(ABSTRACT TRUNCATED AT 250 WORDS)     

丹参素促大鼠心肌细胞及心脏干细胞增殖的作用

目的：大鼠原代心肌细胞及心脏干细胞（CSCs）培养条件的筛选及丹参素（DS-182）对两种细胞体外增殖的影响。方法：对比组织块培养法及差速培养法,选取适宜培养基、消化液浓度、消化时间、差速贴壁时间及心脏成纤维细胞（CFs）抑制剂等因素。分别用MTT比色法、ELISA法及流式细胞术（FCM）检测丹参素对大鼠心肌细胞及CSCs体外增殖的影响。结果：确定DMEM/F12 1∶1培养基、100 ml/L胎牛血清（FBS）、2.5 g/L胰蛋白酶、5 min/次×5次的消化时间及120 min的差速贴壁时间为最适分离纯化条件。MTT比色法检测显示,与空白对照组相比,经浓度为7.8×10-4~1×10-1 mol/L的DS-182处理12、24、48和72 h后,细胞的吸光度（A）值明显增加（P〈0.05）。ELISA法检测显示,与空白对照组相比,经浓度为3.9×10-4~1×10-1 mol/L的DS 182处理24 h后,细胞的A值明显增加（P〈0.05）。FCM检测显示,经浓度为0.1 mol/L的DS-182处理24 h后,c-kit＋的细胞数占细胞总数的比例增加了27.0%。结论：改良了一套较为完整的大鼠心肌细胞及CSCs的分离纯化方法。DS-182在一定浓度下（7.8×10-4~1×10-1 mol/L）对大鼠心肌细胞及CSCs的增殖具有明显的促进作用。     

Synthesis of collagenous proteins by pulmonary type II epithelial cells

We have investigated the production of collagenous proteins by primary cultures of rat lung epithelial cells (type II pneumocytes). Three major bacterial collagenase-sensitive chains were synthesized and secreted into the medium between 12 and 36 h of culture. Two of the chains comigrated on sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis (SDS-PAGE) with radiolabeled type IV procollagen (PC) chains isolated from adult rat lung (Mr = 185,000 and 170,000 after reduction) and were coprecipitated with monospecific antibodies to type IV collagen. Cyanogen bromide (CNBr) peptide maps of the chromatographically purified chains were identical to maps of rat lung type IV PC, and confirmed the identity of these chains as pro alpha 1(IV) and pro alpha 2(IV). Type IV PC was the major high molecular weight collagen in the cell layer, and a fraction of the newly synthesized type IV PC was selectively deposited on the substratum together with newly synthesized fibronectin. Type II cells also secreted a low molecular weight, non-disulfide-bonded, collagenase-sensitive protein (Mr = 19,000, collagen standards; Mr = 26,000, globular standards). The protein coeluted with type IV PC from DEAE-cellulose but was resolved from native type IV on CM-cellulose. The protein was not precipitated with polyclonal antibodies to type IV collagen or rat surfactant apoprotein. These studies further demonstrate the heterogeneity of collagenous macro-molecules synthesized by lung epithelial cells in vitro. We suggest that interactions between pneumocyte-derived fibronectin and type IV procollagen contribute to the formation of the epithelial basement membrane and to the attachment of these cells in normal or injured lung.     

细胞外基质促日本血吸虫培养细胞贴壁作用的研究

目的　研究肝、肺生物基质及鼠尾胶三种细胞外基质 (ECM )对日本血吸虫培养细胞的促贴壁作用。方法　灌注法获取 2 1d虫龄的日本血吸虫虫体 ,冷消化法制备细胞悬液 ,将密度为 2× 10 6/ml的细胞悬液分别接种于均匀铺敷三种基质及未铺敷基质 (对照 )的各组培养瓶中进行培养 ,定时计数贴壁细胞数 ,计算贴壁率。结果　随着培养时间从 8h— 4 8h ,各组日本血吸虫细胞的贴壁率逐渐增高 ,4 8h后下降。同一培养时间 ,基质不同 ,细胞的贴壁率不同。培养 4 8h时细胞的贴壁率 ,鼠尾胶组、肝与肺基质组分别为 6 3 2 7%、4 8 95 %、4 5 36 % ;统计学处理发现 ,它们之间差异显著 ;鼠尾胶组与肝、肺基质组比较 ,p <0 0 1;肝与肺基质组之间比较 ,p <0 0 5 ;对照组与各基质组比较 ,均具显著差异 (p <0 0 1)。 结论　ECM对日本血吸虫培养细胞具有明显的促贴壁作用。其中 ,鼠尾胶对日本血吸虫细胞的促贴壁作用最强 ,其次是肝生物基质 ;肺生物基质的促贴壁作用相对较弱 ,但也强于对照组 (p <0 0 1)。     

A quantitative assay for the adhesion of alveolar type II cells: application to the study of epithelial-fibroblast interactions

A quantitative assay for the adhesion of rat lung epithelial cells has been developed. Cells are adhered to 96 well tissue culture plates and quantitated by their reaction with monoclonal antibodies to cytokeratin in an enzyme linked assay. Using this technique we have shown that rat lung alveolar Type II cells preferentially adhere to a fibroblast monolayer. There was no preferential adhesion of epithelial cells to fibroblast secreted matrix or to the separate components of alveolar matrix.     

Estrogen synthetase (aromatase) activity in primary culture of human term placental cells: effects of cell preparation, growth medium, and serum on adenosine 3',5'-monophosphate response

The establishment of human term trophoblast cells in culture is dependent on the method of cell preparation, growth medium used, and presence of serum. Using freshly isolated term placental cells, we investigated 1) the effects of two different methods of removal of nontrophoblast cells and two culture media on trophoblast aromatase specific activity, cellular morphology, and hCG secretion over 72 h; and 2) the sensitivity of these hormonal parameters to cAMP added 24 h after the initiation of culture. Under conditions in which aromatase is responsive to cAMP, we studied the effect of removing serum after 24 h in culture with serum. After trypsin digestion of placental villi, isolated trophoblast cells were either treated with ammonium chloride (A) or purified on Percoll density gradients (P) and then grown in monolayer culture with medium 199 plus 10% fetal bovine serum (M) or Dulbecco's Modified Eagle's Medium (DMEM) plus 20% fetal bovine serum (D). Regardless of the method of treatment or growth medium used, aromatase specific activity increased 10- to 15-fold 24 h after plating, continued to increase from 24-48 h, and then decreased (AM) or remained constant (AD, PM, and PD) from 48-72 h. Addition of cAMP significantly increased activity in DMEM-grown cells (PD or AD) at both 48 and 72 h. Aromatase activity in PM cells grown with cAMP increased at 48 h, then decreased to near-control levels by 72 h; however, in AM cells, no response to cAMP was observed relative to control cells. Secretion of hCG was suppressed for the first 48 h in all cultures, but increased by 72 h (greatest increase in AM cultures). cAMP significantly increased hCG secretion 48 h after its addition under all conditions. We further evaluated the response of the Percoll-purified DMEM-grown cells (PD) to cAMP after serum removal at 24 h. Cells deprived of serum showed significantly higher aromatase specific activity over the entire culture period compared with serum-grown cells. Secretion of hCG increased 2- or 3-fold in the presence or absence of serum, respectively, after 72 h in culture. cAMP increased aromatase specific activity by 1.8- and 1.4-fold at 48 h and by 2.5- and 2.4-fold at 72 h in serum-containing and serum-free cells, respectively. The secretion of hCG increased 11- to 14-fold at 72 h under both serum-containing and serum-free conditions, respectively. The results show that cAMP can act as an intracellular messenger in the regulation of both aromatase activity and hCG secretion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Bronchoalveolar lavage fluid from normal rats stimulates DNA synthesis in rat alveolar type II cells

Proliferation of alveolar type II cells after lung injury is important for the restoration of the alveolar epithelium. Bronchoalveolar lavage fluid (BALF) may represent an important source of growth factors for alveolar type II cells. To test this possibility, BALF fluid was collected from normal rats, concentrated 10-fold by Amicon filtration, and tested for its ability to stimulate DNA synthesis in rat alveolar type II cells in primary culture. BALF induced a dose-dependent increase in type II cell DNA synthesis resulting in a 6-fold increase in [3H]thymidine incorporation. Similar doses also stimulated [3H]thymidine incorporation into rat lung fibroblasts by 6- to 8-fold. Removal of pulmonary surface active material by centrifugation did not significantly reduce the stimulatory activity of BALF for type II cells. The stimulation of type II cell DNA synthesis by BALF was reduced by 100% after heating at 100 degrees C for 10 min, and by approximately 80% after reduction with dithiothreitol, and after trypsin treatment. Dialysis of BALF against 1 N acetic acid resulted in a 27% reduction in stimulatory activity. The effect of BALF in promoting type II cell DNA synthesis was more pronounced when tested in the presence of serum, although serum itself has very little effect on type II cell DNA synthesis. When BALF was tested in combination with other substances that stimulate type II cell DNA synthesis (cholera toxin, insulin, epidermal growth factor, and acidic fibroblast growth factor), additive effects or greater were observed. When BALF was chromatographed over Sephadex G150, the activity eluted with an apparent molecular weight of 100 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional heterogeneity in somatotrophs isolated from the rat anterior pituitary.

Somatotrophs in suspensions of anterior pituitary cells from adult male rats can be separated into 2 fractions by density gradient centrifugation. In addition to their different densities, somatotrophs in these 2 fractions can be distinguished morphologically by their staining characteristics and ultrastructure. Somatotrophs of lesser density (type I; approximately 1.068 g/cm3) have fewer secretory granules and a more extensive Golgi apparatus than the somatotrophs of greater density (type II; approximately 1.073 g/cm3). Responsiveness of type I and type II cells to secretory agents (i.e., dibutyryl cyclic adenosine monophosphate, somatostatin, thyroxine, and hydrocortisone) was evaluated by GH radioimmunoassay. Type I cells were consistently more responsive (% GH release) than type II cells. During 7 days in culture, type I cells produced more (approximately 200%) GH than they initially contained, whereas type II cells did not show evidence of increased GH production. Hydrocortisone significantly stimulated GH production in type I, but not type II cells. These results support the hypothesis that at least 2 functionally distinct populations of somatotrophs are present in the anterior pituitary gland of the adult male rat.     

Estrogen synthetase activity in human term placental cells in monolayer culture

Human term placental cells, isolated by trypsin treatment, were grown in culture with medium 199 and 10% fetal bovine serum for up to 1 week. Aromatase specific activity (+/-SE) of freshly isolated cells was low (0.63 +/- 0.04 pmol/min X mg protein; n = 15) compared to that of placental tissue before trypsin treatment (21.30 +/- 0.40 pmol/min X mg protein; n = 6). This activity in attached cells increased 10-fold 24 h after plating (6.32 +/- 0.75 pmol/min X mg protein; n = 19) and continued to increase up to 72-96 h (14.78 +/- 1.09 pmol/min X mg protein; n = 13) before declining to 6.50 +/- 1.40 pmol/min X mg protein (n = 4) after 120 h. The functional activity of the cells was assessed by daily measurements of hCG secretion into the medium. Secretion of hCG was maintained at about 0.3 micrograms/flask up to 48 h in culture, then rose rapidly to about 6.2 micrograms/flask from 96-168 h. The addition of 1 mM (Bu)2cAMP plus 1 mM theophylline to the culture medium 24 h after plating stimulated hCG secretion 7- to 8-fold relative to that by control cells, had no effect on aromatase specific activity 24 h after its addition, but decreased aromatase activity after 48 h. Freshly prepared cells were primarily (approximately 80%) mononucleated. With time in culture, the number and size of the multinucleated cells increased drastically until they accounted for virtually all of the cellular material in culture at 72 h. These morphological and functional changes in hCG secretion and aromatase activity suggest that trypsin-isolated cytotrophoblast cells differentiated in culture to form syncytiotrophoblast cells.     

An improved method for isolating type II cells in high yield and purity

A method has been developed for isolating alveolar type II cells by digesting lung tissue with elastase and "panning" the resultant cell suspension on plates coated with IgG. This method provides both high yield and purity of type II cells. In 50 experiments with rats, we obtained 35 +/- 11 X 10(6) cells/rat, 89 +/- 4% of which were type II cells (mean +/- SD). Type II cells isolated by "panning" adhered more rapidly and completely in tissue culture than did cells isolated by centrifugation over discontinuous density gradients of metrizamide. The "panning" method is superior to other methods for isolating type II cells in that it provides a population of type II cells of both high yield and high purity. The method is fast, reproducible, and easily adaptable to isolating type II cells from species other than rats.     

三种培养原代支气管成纤维细胞方法的比较

目的 探讨建立适用于SD大鼠支气管成纤维细胞原代培养的方法.方法 取重量为150~180 g的SD雄性大鼠的支气管组织,采用Ⅰ型胶原酶、木瓜蛋白酶联合消化+组织块黏附法、胰酶+胶原酶联合消化(双酶消化法)+组织块黏附法、组织块黏附法进行原代支气管成纤维细胞的培养.利用镜下观察细胞的生长特点;免疫荧光染色法鉴定细胞的类型及纯度;细胞的增殖活性用CCK-8检测并绘制生长曲线.结果 酶消化+组织块黏附法培养使细胞游出速度更快、细胞数量产生更多.组织块黏附法培养的细胞爬出相对缓慢、培养周期更长、获得的细胞数量相对较少.CCK8增殖曲线呈S形.培养48 h,经酶消化法分离得到的成纤维细胞已经游出,约有60%以上细胞已经贴壁,培养6~7 d可传代.组织块贴壁法培养5 d时,细胞开始从组织块边缘缓慢爬出,随后从组织块周围延伸长,14 d左右可传代.使用酶消化法培养的支气管成纤维细胞中可混有杂细胞团,为提高成纤维细胞的纯度可通过差速贴壁法纯化.结论 双酶消化法+组织块黏附法可以更高效、快速的分离和获取支气管成纤维细胞,为研究慢性阻塞性肺疾病(COPD)气道重塑提供了充足的种子细胞.     

Stimulation of DNA synthesis in cultured rat alveolar type II cells

Restoration of the alveolar epithelium after injury is thought to be dependent on the proliferation of alveolar type II cells. To understand the factors that may be involved in promoting type II cell proliferation in vivo, we determined the effect of potential mitogens and culture substrata on DNA synthesis in rat alveolar type II cells in primary culture. Type II cells cultured in basal medium containing 10% fetal bovine serum (FBS) exhibited essentially no DNA synthesis. Factors that stimulated 3H-thymidine incorporation included cholera toxin, epidermal growth factor, and rat serum. The greatest degree of stimulation was achieved by plating type II cells on an extracellular matrix prepared from bovine corneal endothelial cells and then by culturing the pneumocytes in medium containing rat serum, cholera toxin, insulin, and epidermal growth factor. Under conditions of stimulation of 3H-thymidine incorporation there was an increased DNA content per culture dish but no increase in cell number. The ability of various culture conditions to promote DNA synthesis in type II cells was verified by autoradiography. Type II cells were identified by the presence of cytoplasmic inclusions, which were visualized by tannic acid staining before autoradiography. These results demonstrate the importance of soluble factors and culture substratum in stimulating DNA synthesis in rat alveolar type II cells in primary culture.     

Isolation and culture of human alveolar type II epithelial cells. Characterization of their phospholipid secretion

Alveolar type II epithelial cells have been isolated previously from rodents but not from humans. The aims of this study were to isolate and culture alveolar type II cells from human lungs, and to study their phospholipid secretion. Lung tissue was obtained from 13 patients after lobectomy or pneumonectomy. Type II cells were isolated by a modification of our method for isolating rat type II cells with porcine pancreatic elastase and discontinuous metrizamide density gradients. Cells were purified further by differential adherence and they were then cultured on an extracellular matrix prepared from bovine corneal endothelial cells. We obtained 1.3 to 4.8 X 10(6) cells per gram of tissue with cell viability on the day of isolation of 96 +/- 1% (mean +/- SE, n = 10). Type II cell purity, assessed by Papanicolaou stain on Day 1 in culture, was 84 +/- 4% (mean +/- SE) in 9 of the 13 experiments. In the remaining 4 experiments, the purity was less than 50%. Electron microscopy showed well-preserved ultrastructure. The cells, radiolabeled with 32Pi, secreted the phospholipids characteristic of pulmonary surfactant, primarily phosphatidylcholine (68%) and phosphatidylglycerol (12%). Phospholipid secretion was stimulated by tetradecanoyl phorbol acetate (4.5 times baseline) and by the beta adrenergic agonist terbutaline (2.0 times baseline), but not by the cholinergic agonist carbamylcholine chloride. In summary, human alveolar type II epithelial cells can be isolated from resected lung and maintained in culture, and they secrete the phospholipids characteristic of pulmonary surface active material.     

Inhibin production by sertoli cell cultures

The production of inhibin by cultures of Sertoli cells from 21-day-old rats was assessed by the use of an in vitro bioassay using rat pituitary cells in culture. Sertoli cell culture media (SCCM) caused a dose-dependent suppression of the pituitary cell FSH content which was parallel with that of an ovine testis lymph preparation used as an inhibin standard. SCCM also caused a dose-dependent inhibition of FSH secreted by pituitary cells in response to 10 nM GnRH stimulation. The FSH-inhibitory activity in SCCM was destroyed by heat or trypsin digestion and could not be attributable to the steroid content of the medium, since ether extraction caused no change in the inhibitory activity.The inhibin activity in SCCM was not due to cytotoxicity in the bioassay, since the LH cell content was unchanged and the media produced no change in the release of ⁵¹Cr from labelled pituitary cells, a parameter which has been shown to be a useful test of cytotoxicity. Sertoli cell cultures produced inhibin for the 8-day duration of the cultures. The amount of inhibin produced was proportional to the number of Sertoli cells initially plated. If foetal calf serum was included for more than the initial 48 h, the spent medium caused toxic effects in the pituitary cells as evidenced by an increase in ⁵¹Cr release from ⁵¹Cr-labelled pituitary cells. Similar toxic effects were found if the lyophilized spent media contained cellular debris. A dose-dependent increase in inhibin activity was observed in the presence of graded doses of FSH (0.05–5 μg/ml NIH-FSH-S13).