Optimal conditions for cryopreservation of primary chicken embryo kidney cells with dimethyl sulfoxide

Conditions were evaluated for optimum cryopreservation of primary chicken embryo kidney (CEK) cells. The recovery of viable CEK cells was best (50.8% viability) when the concentration of dimethyl sulfoxide (DMSO) in the freezing medium was 20% (v/v). The viability of primary CEK cells was not influenced by the concentration of calf serum in the freezing medium, the duration of storage at −70°C before storage in liquid nitrogen, cell concentration, or the method of addition or dilution of DMSO. Thawed cells recovered and grew in complete growth medium similarly to cells freshly isolated from kidney, and influenza viruses produced plaques in the monolayer. The cryopreservation procedures described here may facilitate maintenance of a standard stock of primary CEK cells for laboratories where preparation of primary CEK cells is not an option.     

A method for the isolation and serial propagation of keratinocytes,endothelial cells,and fibroblasts from a single punch biopsy of human skin

Summary When multiple types of cells from normal and diseased human skin are required, techniques to isolate cells from small skin biopsies would facilitate experimental studies. The purpose of this investigation was to develop a method for the isolation and propagation of three major cell types (keratinocytes, microvascular endothelial cells, and fibroblasts) from a 4-mm punch biopsy of human skin. To isolate and propagate keratinocytes from a punch biopsy, the epidermis was separated from the dermis by treatment with dispase. Keratinocytes were dissociated from the epidermis by trypsin and plated on a collagen-coated tissue culture petri dish. A combination of two commercial media (Serum-Free Medium and Medium 154) provided optimal growth conditions. To isolate and propagate microvascular endothelial cells from the dermis, cells were released following dispase incubation and plated on a gelatin-coated tissue culture dish. Supplementation of a standard growth medium with a medium conditioned by mouse 3T3 cells was required for the establishment and growth of these cells. Epithelioid endothelial cells were separated from spindle-shaped endothelial cells and from dendritic cells by selective attachment toUlex europeus agglutinin I-coated paramagnetic beads. To establish fibroblasts, dermal explants depleted of keratinocytes and endothelial cells were attached to plastic by centrifugation, and fibroblasts were obtained by explant culture and grown in Dulbecco’s modified Eagle’s medium (DMEM) containing fetal bovine serum (FBS). Using these isolation methods and growth conditions, two confluent T-75 flasks of keratinocytes, one confluent T-25 flask of purified endothelial cells, and one confluent T-25 flask of fibroblasts could be routinely obtained from a 4-mm punch biopsy of human skin. This method should prove useful in studies of human skin where three cell types must be grown in sufficient quantities for molecular and biochemical analysis.     

Cell survival after cryopreservation of dissociated testicular cells from feline species

Testicular cell suspension (TCS) can be cryopreserved for male germ-line preservation and fertility restoration. We aimed to validate a cryopreservation protocol for TCS of domestic cat to be applied in endangered felids species. Testis tissue from adult domestic cats was enzymatically dissociated and spermatogenic cells were enriched. The resulting TCS was diluted in 7.5% or 15% Me2SO based medium. Slow and fast freezing methods were tested. We examined the effects of freezing approaches using two combinations of fluorescent dyes: Calcein-AM with Propidium iodide (C/PI) and SYBR14 with Propidium iodide (S/PI). Ploidy analysis of domestic cat fresh TCS revealed that the majority of testicular cells were haploid cells. Based on microscopic observation, two size populations (12.3 ± 2.3 μm and 20.5 ± 4 μm in diameter) were identified and presumed to be mainly spermatids and spermatocytes, respectively. Both evaluation methods proved higher viability of aggregated cells before and after cryopreservation compared with single cells, and superiority of low concentration of Me2SO (7.5%) in association with slow freezing to preserve viability of testicular cells. However, S/PI resulted in a more precise evaluation compared with the C/PI method. The combination of 7.5% Me2SO-based medium with slow freezing yielded post thaw viability of S/PI labeled aggregated (49.8 ± 20%) and single cells (31.5 ± 8.1%). Comparable results were achieved using testes of a Cheetah and an Asiatic golden cat. In conclusion, TCS from domestic cat can be successfully cryopreserved and has the potential to support fertility restoration of endangered felids species.     

Protection of osteoblastic cells from freeze/thaw cycle-induced oxidative stress by green tea polyphenol

Green tea polyphenol (GTP) together with dimethylsulphoxide (DMSO) were added to a freezing solution of osteoblastic cells (rat calvarial osteoblasts and human osteosarcoma cells) exposed to repeated freeze/thaw cycles (FTC) to induce oxidative stress. When cells were subjected to 3 FTCs, freezing medium containing 10% (v/v) DMSO and 500 μg GTP ml⁻¹ significantly (p < 0.05) suppressed cell detachment and growth inhibition by over 63% and protected cell morphology. Furthermore, the alkaline phosphatase activity of osteoblastic cells was appreciably maintained after 2 and 3 FTCs in this mixture. Polyphenols may thus be of use as a cell cryopreservant and be advantageous in such fields as cell transplantation and tissue engineering.     

Medium composition for effective slow freezing of embryonic cell lines derived from marine medaka (Oryzias dancena)

This study was conducted to identify optimal medium composition for freezing Oryzias dancena embryonic cell lines. Different freezing media consisting of various concentration of dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), and trehalose were prepared and long-term cultured embryonic cell line was frozen in each freezing medium by conventional slow freezing program for 7 days. Through measurement of viability and growth of post-thaw cells frozen in each freezing medium, it was determined that optimal composition of three components was 10 % DMSO, 20 % FBS, and 0.1 M trehalose. The post-thaw cells frozen in optimal freezing medium showed similar morphology and growth rate with non-frozen cells. Next, this condition was applied to two different sets of experiment; (1) freezing of the same cells during expanded period (57 days) and (2) freezing of short-term cultured cells from other batches for 7 days. The viability of post-thaw cells was significantly low and comparable in set 1 and 2, respectively, when compared with the result of long term-cultured cells frozen in optimal freezing medium for 7 days and similar morphology and growth rate with non-frozen counterparts were detected in the post-thaw cells from both sets. In conclusion, this study first reports the optimal medium composition for freezing O. dancena embryonic cells, which can contribute to fish species preservation as well as improvement of cell-based biotechnology by providing stable cell storage.     

A method for infection of cultured myogenic cells with rous sarcoma virus using polybrene

Summary A method for efficiently infecting primary myogenic cultures with a temperature-sensitive variant of the Prague strain of Rous sarcoma virus (RSV, tsLA24) to obtain a high yield of transformed myogenic cells is reported. It incorporates the use of an amorphous polymer of polycations, Polybrene, to enhance the absorption of the virus by the muscle cells. In addition, other steps which were shown to be important were a) to allow cell attachment before infection, b) to infect at 35° C in low protein medium, c) to use a density of 1 to 1.5×10⁶ cells/60-mm dish, d) gentle agitation during infection, and e) to minimize the number of passages after infection. The use of the temperature-sensitive virus provided a means of confirming the presence of myogenic cells in transformed cultures. When infected cells were maintained at 35° C (the permissive temperature for virus activity) they exhibited the characteristics of transformed cells. These characteristics included altered cell morphology, the absence of contact-inhibited growth, growth in semisolid medium, and expression of thesrc oncogene. In contrast, not expresssrc and showed normal myogenic development and ultimately formed myotubes. This work was supported by grants from the Australian Research Grants Committee and the Cancer Foundation of Western Australia.     

Evaluation of isolation methods and culture conditions for rat bone marrow mesenchymal stem cells

Bone marrow mesenchymal stem cells (bMSCs) are multipotent and preferred for cell therapy. However, the content of bMSCs is very low. To propagate a large number of primary bMSCs rapidly has become a prerequisite for bMSC study and application. Different methods of isolating and culturing bMSC were used and compared among groups: bMSCs of group A are isolated using direct adherence method and cultured by conventional medium changing; of group B are isolated using direct adherence method and cultured by low volume medium changing; of group C are isolated using density gradient centrifugation and cultured by conventional medium changing; of group D are isolated using density gradient centrifugation and cultured by low volume medium changing. The average population doubling time (PDT), average generation time and the cumulative cell doubling level were calculated for every group. bMSCs cultured with complete medium containing 10, 11 and 15 % FBS were allocated into group a, b and c separatedly. Cell numbers were counted everyday under a microscope, the population doubling level curve was plotted and PDT was calculated. The growth curve of bMSC in group a, b and c was made. Both density gradient centrifugation and direct adherence methods obtained relatively pure bMSCs. A larger quantity of primary bMSCs were obtained by direct adherence. bMSC proliferation was faster when cultured via the low volume medium changing method at a serum concentration of 11 % than the other methods. Isolating bMSC by direct adherence and culturing by low volume medium changing at a serum concentration of 11 % is preferential for bMSC propagation.     

Root organogenesis from single cells released from the root cap of Medicago sp.

Root border cells were isolated from alfalfa seedlings, and incubated in culture medium with growth regulators. Alfalfa seedlings yielded 1500±100 cells per root, and initial viability of the cells was 95±5%. Multiple cell divisions occurred in the border cells within two weeks. Cell clusters transferred to solidified medium containing growth regulators developed into rapidly growing, friable callus. When transferred to growth regulator-free medium, some of the calluses generated normal roots.Abbreviations BRD cells border cells - SHDN Schenk & Hildebrandt salts medium with growth regulators - SHO Schenk & Hildebrandt salts medium without growth regulators - NAA I-naphthaleneacetic acid     

Natural cryoprotectants combinations of l-proline and trehalose for red blood cells cryopreservation

Cryopreservation of red blood cells (RBCs) holds great potential benefits for supplying transfusion timely in emergencies. Currently, glycerol is the main cryoprotectant permitted in clinical therapy for RBCs cryopreservation, but its broad application is limited by the toxicity and complex deglycerolization process. Successful cryopreservation of RBCs using more effective materials should be studied to reduce freezing damage, increase biocompatibility, and save processing time. Herein, a simple protocol using natural cryoprotectants combinations of l-proline and trehalose attains a low degree of hemolysis (11.2 ± 2.73%) after thawing compared to glycerol. Furthermore, the morphology of RBCs and the activities of Na⁺/K⁺-ATPase and Ca²⁺/Mg²⁺-ATPase maintain well. Further mechanism study shows that l-proline plays an important role in decreasing the freezing points and inhibiting the growth of ice crystal by permeating into cells during the freezing process. While trehalose works as an inhibitor of ice growth in the freezing process and ice recrystallization in the thawing process. This simple l-proline & trehalose combinations protocol is a promising method to replace current time-consuming and labor-intensive cryopreservation methods of RBCs.     

Continuous multiplication of rabbit tracheal epithelial cells in a defined,hormone-supplemented medium

Summary An improved Ham’s F12 nutrient medium supplemented with epidermal growth factor (EGF), insulin (INS), and transferrin (TF) was developed for continuous proliferation and clonal growth of primary rabbit tracheal epithelial (TE) cells in culture. The addition of small quantities of fetal bovine serum (FBS) (0.01 to 0.1%) to cultures had little measurable stimulation on TE cell growth and plating efficiency. However, serum levels higher than 0.1% inhibited cell growth and also masked the growth stimulating activities of EGF and INS despite an increase in cell attachment. Under this defined, hormone-supplemented medium, and in the presence of a trace amount of serum (0.01%), 10 to 20% of the protease-dissociated TE cells attached to the culture dish followed by at least four population doublings during 7 to 10 d of culture. Clonal growth occurred at a seeding density of 17 cells/cm² with a plating efficiency of 6 to 8%. Confluent primary cultures could be passaged two to four times by treatment with a 0.1% trypsin-1 mM EDTA solution and a total of 10 to 30 population doublings of in vitro life span were obtained. The epithelial nature of cultured cells was confirmed by indirect immunofluorescent staining with antikeratin antibody as well as by transmission electron microscopy. This study shows that using this improved hormone-supplemented medium, rabbit TE cells can be maintained in culture for extended periods of time without the aid of a fibroblast feeder layer or explant tissue. This system could be useful for the study of cell differentiation of tracheal epithelium.     

Survival at 20° C and cryopreservation of isolated sperm cells from Zea mays pollen grains

Summary The survival of isolated sperm cells from maize pollen grains at 20° C and their cryopreservation were studied by means of the fluorochromatic reaction (FCR) test. An osmotic pressure of 500 mOsmol, a pH of 7 adjusted with MOPS buffer, and the ten-fold dilution of Brewbacker and Kwack salts enabled isolated sperm cells to survive for a maximum of 35 h at 20° C. The addition of 3 mM of calcium chloride or 0.1 % glucose also slightly improved survival. The addition of 1% glutamine gave the best percentage of FCR-positive cells at 20 h. The addition of all of these substances in a unique medium provoked the formation of spermcell aggregates that were FCR positive. With respect to cryopreservaton, 70% of the isolated sperm cells remained FCR positive after freezing at -80° C and quick thawing at 37° C in the selected survival medium.     

Transferrin is an autocrine growth factor secreted by reuber H-35 cells in serum-free culture

Summary We have previously reported that Reuber H-35 rat hepatoma cells secrete an autocrine growth-stimulating activity in serum-free culture. To characterize this activity, conditioned serum-free medium from dense H-35 donor cultures was collected in the absence and presence of [³⁵S]methionine. A 1∶4 dilution of conditioned medium into fresh serum-free medium resulted in an increase in mean H-35 cell numbers per assay dish from 1.59±0.12×10⁵ to 3.35±0.34×10⁵ after 44 h of incubation. Control, unconditioned medium, resulted in significantly (P<0.05) less growth (2.14±0.41×10⁵ cells per dish). Trypsin digestion eliminated the growth-promoting effect of conditioned medium but had no effect on unconditioned medium. Dialysis did not diminish the growth-promoting activity of conditioned medium. The immunoprecipitate of [³⁵S]methionine-containing conditioned medium with antisera against rat serum transferrin contained a dominant radioactive doublet of molecular weight equal to purified rat serum transferrin. A rat transferrin radioimmunoassay was devised and used to quantitate that 29.1±1.2 ng of transferrin was secreted per 10⁶ cells per hour in serum-free culture. Addition of antitransferrin antibody resulted in a significant (P<0.025) decrease in H-35 cell growth after 48 h. Thus, a portion of the autocrine growth-promoting activity secreted by H-35 cells into serum-free culture is due to transferrin. This work was funded by a feasibility grant from the American Diabetes Association, as well as by grants CA 24604-09 and CA 16463-14 from The National Institutes of Health, Bethesda, MD.     

Growth and differentiation of stage 24 limb mesenchyme cells in a serum-free chemically defined medium

A serum-free defined medium which supports the differentiation of chick limb mesenchymal cells has been developed. In this medium, stage 24 embryonic limb mesenchymal cells which are plated at high density (5 x 10(6) cells/35-mm culture dish) differentiate into chondrocytes. Morphologically, these cultures appear only slightly different from those in which the cells are maintained in serum-containing medium. DNA levels and proline incorporation in cultures grown in defined medium are indistinguishable from control cultures. The rate of radiolabeled sulfate incorporation, a monitor of the rate of proteoglycan synthesis, in Day 8 high-density cultures maintained in defined medium is approximately 70-80% of control values. Additionally, growth and differentiation of intermediate-density (2 x 10(6) cells/35-mm culture dish) and low-density (1 x 10(6) cells/35-mm dish) cultures are also supported by this defined medium. The availability of this medium allows exploration of bioactive factors which affect or modulate mesenchymal cell differentiation and subsequent development.     

Quality of embryonic bodies and seeding density effects on neural differentiation of mouse embryonic stem cells

Mouse embryonic stem (ES) cells can be differentiated into neural lineage cells, but the differentiation efficiency remains low. This study revealed two important factors that influence the neural differentiation efficiency of mouse ES cells: the first is the quality of embryonic bodies (EBs); good quality of EBs consistently originated from a suspension culture of 1 × 10⁵ ES cells/ml serum-free chemically defined neural inducing medium and they exhibited a smooth round shape, with a dark central region surrounded by a light band. Such EBs are capable of attaining high neural differentiation efficiency. However, poor quality EBs originated from a suspension culture of 1 × 10⁶ ES cells/ml serum-free chemically defined neural inducing medium and exhibited an irregular shape or adhered to the bottom of the dish; they displayed low neural differentiation efficiency. The second factor is the seeding density of EBs: a low seeding density (5 EBs/cm²) induced cells to differentiate into a more caudalized subtypes compared to the cells obtained from high seeding density (20 EBs/cm²). These findings provided fresh insight into the neural induction of mouse ES cells.     

Use of enzyme-deficient cell lines as feeder layers

Summary Enzyme-deficient cell lines, lacking TK or HPRT and therefore unable to grow in HAT medium, may be used as feeder layers to enhance clonal growth of wild-type cells. Low numbers of wild-type test cells may be plated in HAT medium with 5×10⁵ HAT-sensitive feeder cells per Petri dish. The feeder cells remain attached and metabolizing for 1 to 2 weeks, but ultimately die and detach, leaving colonies of test cells. This feeder layer technique is very simple and flexible and could have wide applicability. This work was a byproduct of a project on fusion and hybridization of marsupial and eutherian cells supported by the Australian Research Grants Committee.     

Rat pleural mesothelial cells in culture

Summary A culture system has been developed for long-term maintenance of rat pleural mesothelial cells. Mesothelial cells were isolated from the parietal pleura of rats and cultured in NCTC 109 medium supplemented with 10% fetal bovine serum. The cell explants attached to the dish and formed a confluent monolayer of polygonal cells within 10 to 15 days. Subcultures were made in the same medium. The mean population doubling time was approximately 30 hr. The ultrastructure of the mesothelial cells in culture was studied by light and electron microscopy and was compared with that of cells obtained from submesothelial components. This work was supported by CEE Grant No. 264 77 6 ENV F.     

Cheese whey: an alternative growth and protective medium for<Emphasis Type="Italic"> Rhizobium loti</Emphasis> cells

Cheese whey (CW)-based growth medium efficiently protects Rhizobium loti cells during freezing and desiccation and can maintain their growth in a manner similar to that of traditional mannitol-based medium (YEM). The cheese-whey-based medium (CW) improved viability when used to re-suspend cell pellets kept at –20 °C and –80 °C and resulted in the survival of over 90% of the cells. Moreover, bacterial pellets obtained from cells grown in CW withstand desiccation better than cells grown in YEM. Survival was over 60% after 30 days at 4 °C. No differences were observed in nodulation efficiency between YEM-grown and CW-grown cells. Fast protein liquid chromatography (FPLC) protocols are presented for total protein profile analyses of sweet and acid cheese whey.In memoriam of Sylvio Cortina Vicepresident of Fundación COREPRO     

Vitronectin enhances adhesion force and t-PA production of weakly adherent 293 cells exposed to a shear stress

The effects of shear stress on the adhesion andproductivity of 293 cells were studied quantitativelyand compared with those of Vero and human liver cells.These cells were cultured in polystyrene dishes byusing shear stress exposing equipment. 50% of 293cells cultured in 2% FBS supplemented medium detachedfrom the dish after 29 h of exposure to a shear stressof 0.10 Pa. On the other hand, 90% of Vero and humanliver cells remained on the dish under the samecondition. Observations with scanning electronmicroscopy about cell adhesion plaques on the surfaceof the dish showed that the area covered withlamellipodia and the number of microspikes for 293cells were found to be less than those of the othercell lines. Several attachment factors, especiallyvitronectin, were found to enhance the number ofmicrospikes and the adhesion force of 293 cells.Almost 100% of 293 cells remained on thevitronectin-coated dish after 40 h under 0.10 Pa ofshear stress. A higher shear stress (greater than 0.10Pa) caused a decrease in tissue plasminogen activator(t-PA) productivity of 293 cells. But 0.03 Pastimulated the t-PA secretion on the non-coated dish.Vitronectin also enhanced the t-PA secretion evenunder 0.10 Pa. These results indicate that theadhesion force of 293 cells is obviously weaker thanthat of the other cell lines, and vitronectin enhancesthe adhesion force and the productivity of 293 cellsexposed to a shear stress.     

Pyruvate regulation of growth and differentiation in primary cultures of rat tracheal epithelial cells

These studies examined the effect of exogenous pyruvate on the growth and differentiation of primary cell cultures of rat tracheal epithelial cells. The cell cultures were derived from outgrowths of tracheal explants, and require pyruvate for survival and growth in the presence of 10% FBS. In pyruvate-supplemented (2 mM) medium, the number of cells attached to the dish increased rapidly, while exfoliation of cells into the medium as well as formation of cornified envelopes were relatively low. The growth response to pyruvate was concentration-dependent in these cell cultures. In the absence of pyruvate, the extent of terminal differentiation to keratinization gradually increased. This was characterized by a cessation of growth after one week, and an increase in exfoliation until all cells had sloughed from the dish. Accompanying these changes was a marked increase in the formation of cornified envelopes. Cells undergoing DNA synthesis were present throughout 2 weeks of culture in pyruvate-deprived medium, even as the total number of cells was diminishing. Several compounds, including other 2-oxocarboxylic acids, were ineffective growth substitutes for pyruvate. These results indicate that the requirement for pyruvate is quite stringent in these cultures and that one way pyruvate promotes the growth of tracheal epithelial cells is by inhibiting terminal differentiation.     

Proliferation of pulmonary endothelial cells: Time-lapse cinematography of growth to confluence and restitution of monolayer after wounding

A fundamental characteristic of vascular endothelium is that it exists as a monolayer, a condition that must be met in both vascular growth and repair. Maintenance of the monolayer is important both for the exchange of nutrients and for interactions between blood solutes and endothelial enzymes and transport systems. We have used time-lapse cinematography to compare proliferative behavior of bovine pulmonary endothelial cells in (1) establisment of a monolayer from a low-density seed (7.5 × 10⁴ cells in a 60 mm dish) and (2) restitution of a confluent monolayer (approx. 2.9 × 10⁶ cells in a 60 mm dish) following a mechanical wound (removal of cells from an area 5 × 15 mm by scraping). Culture 2 was not refed after wounding. In culture 2, approx. 30% of the cells accounted for repopulation (confluence in 40 hr). In culture I, all cells entered into division. Participating cells of culture 2 began division immediately (69 divisions/filmed area in 10 hr, vs. four divisions in culture I). Interdivision times (IDT) were longer and relatively constant in culture I until near confluence; none were < 10 h, whereas in 2, 24% of the IDT's were ≤ 10 hr. Remarkably, IDTs of culture 2 decreased steadily until confluence was re-established. Cell migration in culture 1 was multidirectional while direction of migration in culture 2 was always into the wound area. Mean migration rate (MIG) in culture 2 was related to the site of origin of the cells, those dividing farthest from the unwounded area had fastest MIGs. Neither culture formed more than a single layer of cells. Although the cell kinetics of cultures 1 and 2 differed, the same goal, confluence, was achieved in either case.