An improved method for culture of epidermal keratinocytes from newborn mouse skin

Reproducible isolation and long term culture of epidermal keratinocytes from transgenic mouse lines is critically needed but most techniques have been unsuccessful. In this report we describe in detail a simplified method to isolate putative keratinocyte stem cells from newborn mouse skin and to maintain them for long term in culture. The cell cultures were established by enzymatically separating keratinocytes from newborn mouse skin. For selecting the putative keratinocyte stem cells for culture, the cells are allowed to attach for 10 minutes on a composite matrix made of type I collagen and fibronectin. Unattached cells were discarded and the attached cells were cultured in a defined culture medium containing low Ca²⁺ concentration, 9% FBS, conditioned medium from newborn mouse skin fibroblasts, and EGF. For subculturing, the cells were seeded on tissue culture plastic. The isolated cells showed the typical basal keratinocyte morphology and expressed the epithelial cell specific integrin v6. The expression level of v6 integrin was comparable to human skin keratinocytes. The keratinocytes were also able to differentiate to form an epidermis in an organotypic culture model. By using the described protocol, the keratinocytes from frozen stocks have been subcultured up to 26 times without change in cell viability, proliferation rate or morphology.     

Persistent infection with mouse hepatitis virus 3 in mouse lymphoid cell lines

The sensitivity of mice to mouse hepatitis virus 3 (MHV3) varies according to strain, age, and immune status of the animals. In semisusceptible strains, mice surviving the acute phase of infection develop a chronic disease characterized by the occurrence of paralysis, virus persistence, and immunodeficiency. Persistent MHV3 infections established in vitro in YAC and RDM -4 mouse lymphoid cell lines were characterized by virus production, presence of cytoplasmic viral antigens, and cell lysis. The occurrence of cell "crisis" in YAC cells was manifested by a sharp increase in cell lysis and in the number of fluorescent cells and, concomitantly, by a marked decrease in virus titers. A relationship was observed among the percentage of fluorescent cells, cell lysis, and virus yield and was modulated by renewal of culture media, change in temperature, or inhibition of cellular RNA synthesis. Cell cloning and antibody treatment experiments indicated that viral transmission was performed by viral infection of newly permissive cells produced by the division of uninfected cells in the culture and not by transmission of viral information by infected dividing cells. The biological and biochemical properties of MHV3 variants derived from persistently infected YAC lymphoid cells were characterized. Thermosensitivity and thermolability of cloned viruses originating from persistently infected YAC cells, as well as parent virus suspensions, were studied. A similar heterogeneity was observed when YAC-derived cloned substrains (YAC-MHV3) were compared with parent-derived cloned viruses, indicating that no selection of temperature-sensitive mutants was induced in persistently infected YAC cells. However, the capacity of MHV3 to induce a lethal acute disease when injected into susceptible mice was lost very rapidly. The absence of pathogenicity was related to the induction of a subclinical infection which elicited defense mechanisms. These data suggest, therefore, that MHV3 replication in lymphoid cell lines leads to induction or selection of variants which maintain pathogenicity in vitro but display reduced pathogenic effects in vivo.     

An improved method for detection and titration of antibodies cytophilic for macrophages

A new microtechnique for detection and titration of antibodies cytophilic for homologous macrophages is introduced and experiments designed to determine the optimum conditions for its performance reported. The technique employs a series of small chambers formed on a microscope slide by the application of a plastic film in which holes have been punched. The procedure uses 5 x 10⁴ cells and 25 μl of serum per chamber, and each test can be completed in 2 h. A permanent preparation results which can be examined as and when convenient.     

Host cell resistance to mouse hepatitis virus type 3 is expressed in vitro in macrophages and lymphocytes

Phenotypic expression of in vivo sensitivity to mouse hepatitis virus type 3 (MHV3) was studied in vitro in macrophages and lymphocytes. MHV3 infections were induced in peritoneal exudate (PE), nonadherent spleen (NAS) and thymus (THY) cells from resistant A/J, susceptible C57BL/6 or semisusceptible (C57BL/6xA/J)F1 mice. Differences in cytopathic effect, cell viability and virus titers were found only at 48 hrs postinfection (p.i.). "Carrier state" infections were performed at 48 hrs p.i. by transfer of supernatants of infected cells to newly collected cells originating from the same strain of mice. A passage-dependent restriction of viral replication was detected in vitro and was expressed in PE, NAS and THY cells as a recessive phenotype. No defective-interfering viral particles were involved in the restriction of viral replication. Results obtained with crossed infections and determination of the number of productively infected cells demonstrated that restriction of viral replication in macrophages and lymphoid cells from resistant A/J mice is controlled by a genetically-determined intrinsic cellular mechanism acting principally on the level of production of infectious viral particles by the infected cell.     

一种改良的间歇性低氧细胞模型方法

 目的: 研制细胞氧舱,建立间歇性低氧(intermittent hypoxia,IH)细胞模型并进行验证。方法: 定制细胞实验舱和空气模拟对照舱,根据氧分压-时间曲线设计间歇低氧模式。将人肺腺癌细胞A549随机分为正常对照(Con)组、间歇低氧6 h(6IH)组、间歇低氧9 h(9IH)组、空气模拟对照6 h(6AC)组、空气模拟对照9 h(9AC)组、持续低氧4 h(4SH)组、持续低氧6 h(6SH)组。暴露结束后光镜下观察细胞形态改变,real-time PCR、免疫组化法检测缺氧诱导因子1α(HIF-1α)的mRNA和蛋白表达变化。结果: 该模型间歇低氧模式为5% O₂ 60 min-20% O₂ 30 min,6个循环。与Con组比较,6AC、9AC组为原本细胞形态,6IH、9IH和6SH组部分细胞出现突起、变圆,胞质中出现较多黑色颗粒,细胞边界模糊,而4SH组未见明显异常。与6IH组比较,9IH组HIF-1α的mRNA和蛋白表达量明显增加(P<0.05);6IH和9IH组HIF-1α的mRNA 和蛋白分别高于4SH和6SH组(P<0.05);6AC、9AC组与Con组比较差异不显著。结论: 5% O₂ 60 min-20% O₂ 30 min的间歇性低氧-复氧细胞模式能模拟阻塞性睡眠呼吸暂停低通气综合征病理生理过程,是研究该疾病较理想的细胞模型。     

Bone-marrow derived macrophages as targets for the replication of mouse hepatitis virus type 3

Bone-marrow (BM) derived macrophages are sensitive target cells for replication of mouse hepatitis virus type 3 (MHV3). These cells can be grown in large numbers and the percentage of defined macrophages increased until day 10 when 100% of the cells represented macrophages. MHV3 replicated within these cells to high titers and caused the formation of multi-nucleated giant cells. This effect was seen with very low virus inocula in BM macrophages of C57BL/6 mice that are highly susceptible to in vivo infection with MHV3 whereas macrophages from resistant A/J mice did not show a cytopathic effect at these virus doses. 1000-fold higher virus doses, however, caused the cytopathic effect in macrophages of both C57BL/6 and A/J mice.     

In vitro growth characteristics and heterogeneity of mouse hepatitis virus type 3

Summary Thein vitro virus yield of MHV3 reached 10⁷ PFU/ml in mouse DBT cells infected with a virus suspension in HEPES-buffered medium containing DEAE-dextran. The virus titer was 10⁶ PFU/ml in the presence of 10 µg actinomycin D/ml. MHV3 grown in DBT cells gave three peaks of density (1.10–1.14 g/cm³, 1.18–1.20 g/cm³, and 1.25–1.31 g/cm³) in sucrose gradients. All these peaks retained infectivity.With 2 Figures     

Replication and plaque formation of mouse hepatitis virus (MHV-2) in mouse cell line DBT culture

Archives of Virology -     

An improved method for quantitative culture of Malassezia furfur.

Quantitative culture of Malassezia furfur from clinically healthy skin in 25 individuals was performed with two different methods using contact plates. The best results were obtained when a glucose peptone yeast extract medium, with the addition of milk, Tween-60, glycerol and glycerol monostearate was used. Different techniques for incubation and the reproducibility of this method were evaluated. Incubation can be done in a plastic bag at 32 or 37 degrees C. This new method is simple, the colonies are easy to identify and the counts are high and reliable.     

改良人颅咽管瘤原代细胞培养方法

目的　探索一种改良人颅咽管瘤细胞培养方法。 方法　根据文献报道和自己的经验,改良既往文献报道的颅咽管瘤细胞培养方法,对两种病理类型的人颅咽管瘤细胞进行原代培养,同时对其进行纯化与传代。通过观察细胞镜下形态、细胞免疫组织化学染色以及细胞增殖实验对原代细胞进行鉴定。 结果　改良后的培养方法培养出的颅咽管瘤细胞从组织块中爬出,排列紧密,透亮且折光性强。细胞免疫组化显示,两种病理类型的细胞均为颅咽管瘤细胞。增殖实验显示,细胞在体外仍具有较好的增殖和分裂的能力。 结论　本研究改良了人颅咽管瘤原代细胞的培养方法,具有简单、经济、成功率高等特点,为颅咽管瘤基础研究提供了良好的保证。     

Kupffer and endothelial liver cell damage renders A/J mice susceptible to mouse hepatitis virus type 3

Damage to the Kupffer and endothelial cells of the liver sinusoids induced by the administration of sublethal doses of frog virus 3 (FV 3) renders A/J mice which are genetically resistant to mouse hepatitis virus type 3 (MHV 3) highly susceptible to this virus. Liver histopathology of these animals revealed typical necrotic foci containing MHV 3-specific antigens. FV 3-pretreated mice, after MHV 3 infection, showed higher levels of serum transaminase (GPT) than controls, and MHV 3 replicated more rapidly and to higher titres. Our results bear out the important role of the liver sinusoidal lining in protecting against hepatocyte infection and its direct involvement in the resistance of A/J mice to MHV 3 infection.     

An improved method for determining proteoglycans synthesized by chondrocytes in culture

An improved micro method for measuring sulfated glycosaminoglycans (S-GAG) in chondrocyte cultures using 1,9-Dimethylmethylene Blue (DMB) has been developed. By increasing the protein concentration in the DMB assay a soluble GAG-DMB complex is prolonged. Without bovine serum albumin (BSA) in the phosphate-buffered saline (PBS) medium, the half time for loss of absorbance was 18 min; with 1% BSA-PBS there was no loss of absorbance over this time period. The limit of detection in a 96 well microtiter plate assay was 2 micrograms/ml; for a cuvette assay it was 1 microgram/ml. Collagen, DNA and RNA did not interfere with this assay. Hyaluronate caused an increase in absorbance at 530 nm that was lost by preincubating with Streptomyces hyaluronidase. The increase in absorbance was due to a turbidity change because there was no color shift from 600 to 530 nm but rather a uniform increase in absorbance between 400 to 700 nm. To validate the assay, the S-GAG was measured in conditioned medium from primary bovine articular chondrocyte monolayer cultures. A protein synthesis inhibitor, cycloheximide, blocked proteoglycan synthesis by greater than 90%. A cytokine, Interleukin-1 alpha, caused a dose-dependent decrease in proteoglycan accumulation. Chondroitinase ABC digestion of the chondrocyte conditioned medium completely prevented reactivity with the DMB. By preincubating samples with specific enzymes, different types of S-GAG can be measured with this assay. This assay can be used to measure changes in proteoglycans synthesized by chondrocytes.     

A metabolic inhibition test for titration of antibody to arboviruses adapted to mouse embryo cell culture

Summary Several arboviruses were adapted to cultures of mouse embryo cells. A color test system (metabolic inhibition test) is described in which mouse embryo cell suspensions are applied.There was a fairly good agreement between the results of cross neutralization reactions in the metabolic inhibition test and in the mouse protection test.Approximately 7% of more than 200 serum samples from Dutch military servicemen stationed in Surinam was shown to possess antibody to a group of three immunologically related viruses (Paramaribo, Semliki Forest and Chikungunya) as determined in the metabolic inhibition test.     

Protection of mice against infection with mouse hepatitis virus type 3 by injection of silica

Injection of silica did not brake the resistance against MHV3 conferred to C57BL/6 mice by injection of C. parvum. However, silica itself had a marked protective effect against MHV3 infection that was maximal when injecting 1 mg 2 hrs before virus infection. The protective effect of silica was observed in a number of inbred mouse strains that differ in their relative resistance to MHV3 infection. No viral titers were observed in the spleen and liver of mice which had received MHV3 plus silica, whereas high titers were observed in the virus-infected controls. Injection of silica caused a marked decrease in the number of esterase-positive macrophages in the peritoneal wash-out population, that may be compatible with the possibility that the cause of the protection is the depletion of target cells for the viral infection. This latter effect, however, was short-lived and 24-48 hrs after injection of silica, high numbers of esterase-positive cells were again observed. This may explain why only little protection was observed when silica was administered 2 days before virus infection.     

一种改进的大鼠大脑皮质星形胶质细胞的培养方法

目的:改进大鼠大脑皮质星形胶质细胞的培养方法,从而获取高纯度星形胶质细胞用于神经科学研究。方法:新生大鼠(2~3日龄),75%酒精消毒后断头,于冰上取大脑皮质置于冰冷的D-Hanks液中,去除脑膜和血管,剪碎后用0.25%胰酶消化并分离细胞,以1×10~5/ml的密度接种于预铺0.015%多聚赖氨酸的细胞瓶中培养。细胞纯化采用"差速贴壁法"、"梯度血清法"和"十字手摇法",以获取高纯度星形胶质细胞。通过星形胶质细胞特异性表达蛋白即胶质原纤维酸性蛋白(GFAP)行免疫细胞荧光染色来鉴定细胞纯度。结果:星形胶质细胞纯度大于98%,可多次传代,细胞增殖快,且生长良好。结论:改进的原代星形胶质细胞培养方法,操作简单,细胞纯度高,能满足各种神经科学研究中对高纯度星形胶质细胞培养的实验要求,适宜推广。     

An improved method for elimination of mycoplasmas from cell cultures

Cell lines infected by different species of mycoplasma (Mycoplasma orale, Mycoplasma hominis) were decontaminated by co-culture with human blood monocyte (BM)-derived macrophages and pooled human immunoglobulin preparations. Co-cultures with BM-derived macrophages or murine peritoneal macrophages (PM) alone were not successful. The phenotype of infected cell lines did not differ from that of uninfected cell lines as revealed by morphological, enzymecytochemical, and immunocytochemical analysis.