胎鼠脊髓神经干细胞分离方法的比较

目的：比较机械法和胰酶消化法对胎鼠脊髓源神经干细胞增殖分化的影响。方法：分别用机械法和胰酶消化法分离胎鼠脊髓组织获得神经干细胞,应用台盼蓝检测细胞成活率,用无血清培养技术培养神经干细胞,应用MTT法检测细胞分裂增殖能力．采用免疫细胞化学法鉴定神经干细胞和分化细胞。结果：机械法获得的细胞数量多于胰酶消化法。细胞经过培养其增殖能力机械法略强于胰酶消化法,但无统计学意义。培养形成的细胞球Nestin阳性,诱导分化后可见NSE和GFAP阳性细胞。结论：运用机械法比胰酶消化法分离胎鼠脊髓组织获得神经干细胞方法简单,容易操作,经过培养细胞增殖能力较强。并可提供健康的细胞来源。     

Spontaneous and lectin-induced redistribution of cell surface receptors on embryonic chick neural retina cells.

The mobility of plant lectin receptors in the plane of the membrane is examined for cells prepared from embryonic chick neural retinas by a variety of procedures. Cells liberated from the intact tissue by trypsin treatment followed by mechanical dissociation are able to redistribute their receptors into 'caps' both spontaneously and in the presence of a multivalent lectin. These cells, dispersed by trypsinization, upon repair in culture for a suitable period of time lose their ability to redistribute lectin receptors. Cells dispersed by mechanical means without prior trypsin treatment are unable to undergo 'cap' formation. In addition, cells within intact tissues are also unable to redistribute their lectin receptors into 'caps.' Based on these observations we propose that within solid tissues which have assumed their characteristic architecture, cell surfaces are immobilized, and that this phenomenon may be a critical parameter in determining the potential of a cell to undergo morphogenetic rearrangements.     

Flattening,movement and control of division of epithelial-like cells

The kinetics of cell division and movement in four epithelial-like cell lines, grown in continuously perfused culture medium, were studied by time-lapse cinemicrography. One line exhibited “contact regulation of cell division,” so that the rate of mitosis per cell decreased steadily as population density increased. In the other three lines mitosis was not controlled as a function of population density until the cells became very crowded. An explanation for this difference was sought in terms of the hypothesis that the rate of division depends on the area of the cell membrane. Cells of the contact-regulated line flattened uniformly on the substrate. Their motility was restrained by adhesion between their borders. As they crowded together, contact inhibition of cell overlap caused a steady decrease in average surface area per cell. All three of the non-controlled lines also had contact inhibition of overlap. Cells of two of them flattened on the substrate; but these cells had little mutual adhesion and were highly motile, so that they continually changed their shapes. The areas of their cell membranes were therefore not subject to a restraint that could control the rate of division. Cells of the fourth line remained rounded or only slightly flattened during culture growth, so that no change in cell membrane area occurred that could change the rate of division.     

Effect of cell surface trypsinization on ethanolamine base exchange enzymatic activity of astrocyte primary cultures and derived spontaneously transformed cell lines

Trypsinization of neonatal rat astrocyte primary cultures (normal cells) inhibited the activity of ethanolamine base exchange enzyme (EBEE) by 80%, whereas ethanolamine phosphotransferase (EPT) and choline base exchange (CBEE) enzymatic activities were not affected; subcellular fractionation demonstrated that trypsin treatment affected the intracellular EBEE activity. During trypsinization the enzyme was not taken up by cultured astrocytes but the cell surface was affected. In contrast, the same treatment did not alter EPT, CBEE and EBEE activities of spontaneously transformed cell lines derived from the primary cultures. However, treatment of the transformed cells with db-cAMP prior to trypsin, restored the pattern found in the primary culture, i.e. only EBEE activity was affected. These data suggest that a relationship exists between cell surface organization and intracellular EBEE activity in a culture system which possesses the property to control its own cell division or/and differentiation.     

猪耳皮肤成纤维细胞的培养

本研究以猪耳皮组织为材料,采用胰蛋白酶冷热处理结合法成功地分离和培养了猪耳皮肤成纤维细胞。此方法的细胞存活率相对于胰蛋白酶热处理法较高。传代细胞与原代细胞的形态和生长速度均相似。传代细胞未检测到凋亡现象。细胞已传至15代以上,染色体倍性正常,说明我们所建立的胰蛋白酶冷热处理结合法可以快速有效的分离和培养猪耳皮肤成纤维细胞,并且能稳定的进行传代培养。     

Multistage continuous culture to examine secondary metabolite formation in plant cells: Phenolics from Nicotiana tabacum

The feasibility of operating a multistage continuous culture of plant cells was demonstrated for Nicotiana tabacum. Cells in the second stage of a two-stage chemostat were morphologically distinct from cells in the first stage or cells in a single-stage unit with a holding time equal to the combined holding times in the two-stage system. Cells in the second stage produced much higher levels of phenolics per unit weight of cells than cells in either the first-stage or single-stage unit. The steady-state was reproduced. When a glucose side stream was fed to the second stage, an increase in apparent cell division was observed with a simultaneous decrease in phenolics productivity. When the toxic precursor phenylalanine was pulsed into the reactor, the quantity of biomass decreased temporarily while phenolic productivity increased. These experiments demonstrate that multistage continuous culture may be useful in increasing secondary metabolite formation in cells and in exploring mechanisms controlling secondary metabolite formation.     

Fluctuations in buoyant density during the cell cycle of Escherichia coli K12: significance for the preparation of synchronous cultures by age selection.

The buoyant densities of Escherichia coli K12 were investigated by isopycnic centrifugation in gradients of colloidal silica (Ludox) and polyvinylpyrrolidone. Bacteria from an exponential culture in a defined medium supplemented with hydrolysed casein banded at densities between 1-060 and 1-115 g ml-1; the mean density was 1-081 g ml-1. At the higher densities, two populations of cells were present: smaller cells were approximately twice as numerous as, and half the modal volume of, the population of larger cells. A homogeneous population of cells of intermediate volume equilibrated in the least dense region of the density band. Synchronous cultures were established by inoculating cells selected from the most or least dense regions of the band into spent growth medium. The results are consistent with a fluctuation between maximal density at cell birth and division, and minimal density near the middle of the cell cycle. In synchronous cultures prepared by continuous-flow age selection, the first division occurred after a period that was significantly shorter than the length of subsequent cell cycles. Cells selected by this procedure were of similar mean density to those in the exponential culture but were more homogeneous with respect to size. The possibility that the smallest (and densest) cells in an exponential culture are retained in the rotor, and are thus excluded from the synchronous culture, is discussed.     

Standardization of Human Diploid Fibroblast Cultivation: Trypsinization Procedure

Human embryonic diploid lung fibroblasts were used to examine the influence of the trypsinizing procedure on the growth and adsorption of these cells. The best buffer for trypsinizing these cells was Hanks balanced salt solution containing 0.5% lactalbumin hydrolysate. Trypsinizing cells at 4 C gave better growth results than trypsinization at higher temperatures. The presence of antibiotics in the trypsin buffer increased the longevity of the cells. Cells initially trypsinized from tissue in phosphate-buffered saline without Ca(2+) or Mg(2+) and 0.33 m sucrose plus 10(-3)m Mg(2+) gave rise to better subsequent growth and adsorption than cells from tissue trypsinized in other buffers.     

Glutamine synthetase and energy metabolism enzymes in cultured chick glial cells: Modulation by dibutyryl cyclic AMP,hydrocortisone, and trypsinization

Modifications induced by dibutyryl cyclic AMP (diBcAMP) and hydrocortisone in the energy metabolism of chick astroblasts in culture have been investigated. DiBcAMP does not modify the levels of enolase, malate dehydrogenase (MDH), total lactate dehydrogenase (LDH) and glutamine synthetase (GS) activities in these cultured glial cells. However, these cells can be sensitized to the nucleotide analog by trypsinization before seeding. The phenomenon affects specifically GS activity and the synthesis, with an inhibitory effect, of the H subunit of LDH. Addition of hydrocortisone to the culture medium stimulates MDH and GS activities of the cells; trypsinization accentuates the stimulatory effect on GS. This hormone also modifies the synthesis of H and M subunits of LDH in a positive and negative way respectively. The phenomenon is increased by trypsin treatment. The present studies indicate clearly that hydrocortisone generates in cultured chick glial cells metabolic modifications qualitatively different from those obtained by diBcAMP. It is suggested that trypsin treatment, by altering some protein constituents of the cell surface, modifies the adhesiveness of different cell types present in the cell suspension after dissociation of the brain and thus leads to select, in culture, a specific astroglial subpopulation.     

Serum, trypsin, and cell shape but not cell-to-cell contact influence the X-ray sensitivity of Chinese hamster V79 cells in monolayers and in spheroids

Nutrient concentration in the growth medium and trypsin affect cellular radiosensitivity in a manner that is related to cell shape (Reddy, Stevenson, and Lange, Int. J. Radiat. Biol. 55, 105-117 (1989); Reddy and Lange, Radiat. Res. 119, 338-347 (1989]. Hence we hypothesized that the concentration of serum in the medium could influence the X-ray sensitivity of cells and that the spread cells in monolayers and round cells in spheroids may differ in their response to the radiosensitizing effect of trypsin. We compared the X-ray sensitivity of monolayer and spheroid cells grown for 19 +/- 1 h in MEM supplemented with 5 or 15% serum. Cells were trypsinized and plated either immediately before, or 2.5 +/- 0.5 h after, irradiation and incubation for repair in situ. Survival of cells in monolayers and in spheroids was higher in MEM with 5% serum than with 15% serum. Trypsin treatment affected the shape and radiosensitivity of cells in monolayers but not in spheroids. When all cells were grown in the same serum concentration and a 2.5-h postirradiation incubation was allowed prior to trypsinization, the X-ray sensitivity of cells in spheroids was greater than that of cells in monolayers. The survival of cells in spheroids became equal to that of monolayer cells when cells in spheroids were converted to monolayers by placing them in 25-cm2 flasks and allowing them 3 h to attach and spread. Cell cycle distributions were nearly the same in monolayers and spheroids cultured in MEM with 5 or 15% serum. We conclude that: (1) serum concentration in the growth medium and trypsin do appear to contribute to the differences in the radiosensitivity of spheroids and monolayer V79 cells; (2) these differences are associated with changes in cell morphology.     

牛成纤维细胞的分离与体外培养

研究了牛胎儿和成年牛皮肤组织成纤维细胞的分离、培养、纯化方法和生长特征。通过组织块贴壁培养和分离单细胞接种培养均能获得原代牛皮肤细胞。用2.5 g/L胰蛋白酶+1mmol/L EDTA和5 g/L胶原酶I联合消化牛皮肤组织较2.5 g/L胰蛋白酶+1 mmol/L EDTA消化,得到更多的单个细胞,两者之间差异极显著(P<0.01),但其死细胞比率却有较大升高;2.5 g/L胰蛋白酶+1 mmol/L EDTA消化牛胎儿组织得到的单细胞数显著高于皮肤组织消化后得到的细胞数(P<0.01),死细胞比率也高于同种酶消化的皮肤组织。分离纯化的胎儿和皮肤成纤维细胞的生长曲线都正常且相似。2.5 g/L胰蛋白酶+1 mmol/L EDTA消化贴壁细胞后死细胞率明显高于用0.5g/L胰蛋白酶+0.53 mmol/L EDTA消化的细胞(P<0.05);培养24 h后细胞贴壁率前者要明显低于后者(P<0.05)。用0.5 g/L胰蛋白酶轻度消化混杂生长的成纤维细胞和上皮样细胞,经过反复贴壁传代2～3代,可得到较纯的成纤维细胞。     

Multiplication of human diploid fibroblasts in a synthetic medium supplemented with EGF, insulin, and dexamethasone

Substantial multiplication of human diploid fibroblasts (HDF) has been obtained in medium MCDB 108 supplemented with epidermal growth factor (EGF), insulin, and dexamethasone (DEX). Growth rate is somewhat slower than in serum-supplemented medium. However, large wellformed colonies can be obtained in 14 days, and sequential monolayer subculture is possible up to a total of about ten population doublings. A basal medium that has been optimized specifically for HDF is essential for such multiplication. In addition, polylysine-coated culture surfaces, low temperature trypsinization, and careful removal or neutralization of residual trypsin are also needed. The culture system contains no deliberately-added undefined components, and is chemically defined except for possible roles of contaminants in the materials that are used for its preparation.     

Reculture of Electrofocused Foetal Rabbit Skin Cells

Cells from a primary culture of 30 day-old foetal rabbit skin were electrofocused and recultured. Certain fractions (pH 10-3) grew best on reculture. Electrofocused cells formed monolayers in six weeks. The focused cells were dormant for a period preceding growth. Increased cell density and cell contact of recultured cells occurred when they were exposed to millipore filtered media from unfocused cells. Primary culture cells exposed to 1 mw L -aspartic acid, L -ornithine, or L -lysine in culture media, when treated with trypsin and electrofocused indicated different profiles of pH maxima. These recultured cells differed from the primary culture cells in morphology and growth rate.     

Molecular changes in cell surface membranes resulting from trypsinization of sarcoma 180 tumor cells

Sarcoma-180 tumor cells in culture or grown as an ascites form in the CD-1 mouse have been subjected to mild trypsinization procedures in order to study morphological and molecular changes resulting from proteolysis. The cells attached to a substratum become rounded within 20 min and most undergo cell division, but they do not detach from the substratum. Removal of trypsin permits the cells to go back to their original spindle shape over an 8–20 h period.Surface membranes were isolated from trypsinized ascites and cultured cells and subjected to dodecyl sulfate-acrylamide gel electrophoresis. Both cell types showed the same two kinds of changes in electrophoretic patterns. First, there was a loss of glycoproteins from both cell types, even though they show different complements of cell surface glycoproteins. Second, there is a loss of high molecular weight polypeptides, which have previously been suggested to play a role in membrane stabilization and cell shape. These results further implicate these polypeptides in the control of cell morphology and offer circumstantial evidence for transmembrane interactions of surface glycoproteins with the high molecular weight polypeptides as a factor in controlling cell morphology.     

Growth properties and biochemical characterization of mouse Schwann cells cultured in vitro

Purified secondary cultures of mouse Schwann cells (less than 5% fibroblast contamination) have been obtained by taking advantage of the differential adhesion of Schwann cells and fibroblasts during trypsinization. The growth properties of the purified subcultures changed with time in culture. Cells passaged after 5 days in vitro (DIV) divided rapidly (doubling time 22 h), whereas cells that had been in vitro for longer periods progressively decreased their growth rate, becoming quiescent after 20 or more days. Schwann cells lacked the Thy 1.2 surface antigen, but were positively stained with antigalactocerebroside antibodies after prefixation. Biochemical analyses showed Schwann cells to be enriched in the activities of enzymes characteristic of the myelin-forming cells: 2′3′-cyclic nucleotide 3′-phosphodiesterase (CNP), cerebroside sulfotransferase (CST) and UDP-galactose: ceramide galactosyltransferase (CGalT).     

Molecular changes in cell surface membranes resulting from trypsinization of sarcoma 180 tumor cells.

Sarcoma-180 tumor cells in culture or grown as an ascites form in the CD-1 mouse have been subjected to mild trypsinization procedures in order to study morphological and molecular changes resulting from proteolysis. The cells attached to a substratum become rounded within 20 min. and most undergo cell division, but they do not detach from the substratum. Removal of trypsin permits the cells to go back to their original spindle shape over an 8-20 h period. Surface membranes were isolated from trypsinized ascites and cultured cells and subjected to dodecyl sulfate-acrylamide gel electrophoresis. Both cell types showed the same two kinds of changes in electrophoretic patterns. First, there was a loss of glycoproteins from both cell types even though they show different complements of cell surface glycoproteins. Second, there is a loss of high molecular weight polypeptides, which have previously been suggested to play a role in membrane stabilization and cell shape. These results further implicate these polypeptides in the control of cell morphology and offer circumstanital evidence for transmembrane interactions of surface glycoproteins with the high molecular weight polypeptides as a factor in controlling cell morphology.     

Analysis of rat adrenal cells in primary culture separated by velocity sedimentation

Summary Cell separation was used to follow the fate of the cortical cells of the adrenal gland in primary culture, and to assess some of the changes that occur as cells adapt to culture conditions. Primary cultures of rat adrenal gland were dissociated with trypsin and separated by velocity sedimentation at unit gravity. After two days in culture, cells showed a reproducible sedimentation profile consisting of two classes of cells with mean sedimentation rates of 5.8 and 2.1 mm/h, and a third sedimentation peak consisting mainly of nuclei at 0.5mm/h. All populations continued to incorporate ³H-thymidine in relatively constant proportion throughout the culture period, but the relative number of cells in the 2.1 mm/h peak increased two-fold in the last few days of primary culture. Cells labelled in primary culture, but separated after an additional 5 days in secondary culture had lost proportionately more labelled cells from the 5.8 mm peak. The results suggest that cells of the 2.1 mm peak survive longer in culture in a post-replicative condition.Work reported in this paper was performed while the author was a Research Fellow of the National Cancer Institute of Canada in the laboratory of Dr. N. Auersperg, Cancer Research Centre, University of British Columbia, Vancouver, B.C., Canada     

Cell Dispersal Methods for Increasing Yield from Animal Tissues

An improved method for increasing cell yield from animal tissues is described. The current technique differs from the classical, primarily in that the tissues were perfused with trypsin in situ and cell dispersal was completed by trypsinization either at 4 or 37 C but without prior mincing of the tissue. Increased yields of 75% were obtained. Mechanical damage to tissue by mincing was presumed to be the greatest single source of injury to cells. The adoption of the trypsin perfusion technique reduced the number of animals needed to prepare an equivalent quantity of tissue culture.     

A detachment technique based on the thermophysiologic responses of cultured mesenchymal cells exposed to cold

Trypsinization has generally been used as a technique to detach adherent mesenchymal stromal cells (MSC). However, this technique involves chemical manipulation. This study was designed to identify whether detachment of MSC can be induced by cold without using trypsin. MSC isolated from bone marrow were detached via trypsin or exposed to -20 degrees C for 1, 5 or 10 min at all passages. Compared with trypsinization, exposing MSC to -20 degrees C for 10 min resulted in a significant decrease in MSC number and viability. In conclusion, although detachment of adhered MSC on culture dishes via exposure to cold may allow structurally and functionally intact detached cells, the technique requires improvement of the thermotolerance of MSC.     

Fibronectin delays the fusion of L6 myoblasts

The effect of fibronectin on myogenesis has been studied in vitro. The addition of purified fibronectin to the myogenic cell line L₆ blocks fusion and causes an increase in cell number. The effects of fibronectin could be prevented by the immunoprecipitation of fibronectin from solutions using affinity-purified antifibronectin antibodies. Mild trypsinization of the cells (10 μ/ml trypsin for 20 min) which removes surface fibronectin, causes the rate of fusion to increase when the trypsinization is done just before the cells begin to fuse, day 4 (after the plating of the cells), an inhibition when done on one day earlier, day 3, and has no effect when done after the cells have begun to fuse, day 5. By measuring the binding of rhodamine-labeled antifibronectin antibodies to intact cells, it was found that surface fibronectin increased from day 3 to day 4 and then decreased on day 5. The stimulating effect of trypsin on fusion, therefore, corresponds to the day surface fibronectin reaches a peak. Affinity-purified antifibronectin antibodies were also shown to be capable of enhancing fusion. It is concluded from these results that high levels of fibronectin stimulate events which reduce fusion, whereas the removal of surface fibronectin during critical times either stimulates or reduces fusion.