New approaches to insect tissue culture

Conclusion Current methods of insect cell culture have produced a limited variety of cell types in an ever expanding list of insect cell lines. In developing midgut epithelial cell lines, we found that traditional methods in insect cell culture failed to provide healthy cells from mature tissues. Examination of mammalian cell culture literature for this particular cell type provided the insight required to successfully develop a cell-specific line (Baines et al., 1994). The potential applications for cell-specific lines from insects are numerous. This paper is a compilation of ideas that will hopefully enable other researchers to develop additional cell-specific lines.     

Sensitivity of ovine myometrial tissue to various eicosanoids in vitro

The sensitivity of sheep myometrial tissue to prostaglandin F_2α (PGF_2α), PGE₂, the thromboxane analog U-44069, and leukotrienes C₄ (LTC₄) and LTD₄ was investigated in a superfusion system. Tissues were obtained from eight oophorectomized ewes, with or without pretreatment with estradiol-17β. After equilibration, spontaneous activity was abolished by adding indomethacin to the superfusion fluid. The dose needed to induce a contraction with a peak level of 50% of the median peak level of spontaneous contractions increased from PGE₂ to PGF_2α, U-44069, LTC₄, and LTD₄. The differences between the doses required were significant for all compounds, except between LTC₄ and LTD₄. Estradiol-17β pretreatment caused an increase in the required dose of PGF_2α. The results of this study do not support the hypothesis that leukotrienes are involved in the regulation of myometrial activity.     

Sensitivity of ovarian tumor cells to effector cells generated by various biological response modifiers

The sensitivity of freshly derived human ovarian tumors (FOT) to various allogeneic cytotoxic effector cells stimulated by recombinant interleukin 2 (rIL-2), recombinant interferon alpha 2 (rIFN-alpha 2), OK-432, and concanavalin A was examined using the 51Cr release assay. Peripheral blood lymphocytes (PBL) of normal female donors were used as source of effector cells. Incubation of PBL with these biological response modifiers for 24 h generated effector cells with high natural killer activity, and only 20% (1/5) of the FOT examined were susceptible to lysis. By contrast, 83% (5/6) of the FOT were sensitive to lymphokine-activated killer (LAK) cells generated by rIL-2. OK-432 and concanavalin A activation of PBL also generated cytotoxic cells, though the cytotoxic activity against FOT was much less than that obtained by LAK cells. The addition of OK-432 to LAK culture medium containing rIL-2 generated effector cells with higher cytotoxicity against FOT than cultures with IL-2 alone. However, the addition of rIFN-alpha 2 in LAK culture medium resulted in the generation of effector cells with lower cytotoxicity. The addition of rIL-2, rIFN-alpha 2, or OK-432 to LAK cells during the in vitro cytotoxicity assay had no significant effect. When FOT target cells were pretreated with OK-432 they became more sensitive to LAK than nontreated tumor cells. However, pretreatment with rIL-2 or rIFN-alpha 2 did not influence cytolysis. These results suggest that the generation of LAK cells in vitro using rIL-2 plus OK-432 may be a more effective way to prepare these cells for adoptive immunotherapy in the treatment of ovarian cancer.     

Steroidogenic capabilities of various compartments of rat ovarian follicles in culture

The production of progesterone, estrogen and androgen as well as the metabolism of radiolabelled progesterone by various cellular components of rat ovarian follicles were studied. Granulosa (G), theca (T), recombined granulosa plus theca (G+T) and intact follicular wall (FW) of ovaries from immature rats treated with pregnant mare serum gonadotropin (8 IU) were cultured for 24 h in the presence or absence of [4-¹⁴C]progesterone. The estrogen and androgen accumulation when calculated per follicle was several fold greater in FW than in G,T, or G+T preparations. The conversion of radiolabelled progesterone to its identified C₂₁ catabolites (20α-hydroxy-4-pregnen-3-one and 3α-hydroxy-5α-pregnan-20-one) was significantly lower in FW than in G+T incubations. Conversely, the metabolism of radiolabelled progesterone to androsterone was several fold greater in FW than in G+T incubations. Addition of hydroxyflutamide to FW incubations significantly decreased estrogen production and increased the conversion of radiolabelled progesterone to androsterone. Estrogen production by follicular wall may be enhanced by androgenic stimulation of aromatase activity as well as by a structure-dependent factor(s) of a yet unknown nature, both of which may decrease progesterone catabolism to biologically inactive progestins while promoting progesterone conversion to androgens and eventually to estrogens.     

T-DNA rearrangements due to tissue culture: somaclonal variation in crown gall tissues

After three years of apparent stability in tissue culture, the single cell derived shooty crown gall line sNT1.013 produced a revertant shoot which had switched from non-rooting (Rod⁺) and octopine synthesizing (Ocs⁺) to Rod^- Ocs^-, indicating that in this revertant TL-DNA genes 4 (causing the Rod⁺ trait) and gene 3 (causing the Ocs⁺ trait) had been inactivated. Southern blots revealed that the inactivation of these T-DNA genes was the result of a considerable rearrangement of DNA sequences, accompanied by deletions and possibly also by DNA amplifications. This study for the first time unambiguously proves that foreign genes which have been introduced via Agrobacterium tumefaciens can, at a low frequency, be inactivated after T-DNA integration because of reorganization of T-DNA sequences during tissue culture. This can be considered as an event of somaclonal variation.     

Serial propagation of human ovarian surface epithelium in tissue culture

Most human ovarian cancers are thought to arise in the ovarian surface epithelium (OSE). The precise role of OSE in carcinogenesis has not been defined because no appropriate animal models for the study of this tissue exist and culture of human OSE has been limited to primary outgrowths. In this report, we describe conditions for serial cultivation of normal human OSE. Premenopausal ovarian tissue was obtained at surgery. OSE growth was compared in media MCDB 202, 199 and Waymouth's 752/1 (WM) supplemented with 5, 15, or 25% fetal bovine serum (FBS), with/without 20 ng/ml epidermal growth factor (EGF) and 0.4 micrograms/ml hydrocortisone (HC). The rate and extent of OSE outgrowths from explants in primary culture were greatest in either WM or 199/202 (1:1), supplemented with 15% FBS/EGF/HC. In early passage cultures, cell proliferation was most rapid and extensive in 199/202 with 15% FBS, EGF, and HC. In this medium, OSE cells were subcultured up to 10 times and underwent 20-25 population doublings over 5 weeks. The population doubling time during rapid growth was approximately 48 h. Seeding efficiencies of up to 53% and cloning efficiencies of up to 13% were obtained. Early passage OSE cells reversibly modulated from a slow growing, epithelial, intensely keratin-positive form in 199/202 medium lacking EGF/HC, to a rapidly proliferating, elongate, less keratin-positive form in medium with EGF/HC. OSE cells grown in WM/5-15% FBS were epithelial and near-stationary. Thus, culture conditions have been defined for ovarian carcinogen assays requiring either proliferating or stationary cell populations, and for further studies of the role of OSE in ovarian biology.     

Predicted permeability parameters of human ovarian tissue cells to various cryoprotectants and water

This study presents a generic numerical model to simulate the coupled solute and solvent transport in human ovarian tissue sections during addition and removal of chemical additives or cryoprotective agents (CPA). The model accounts for the axial and radial diffusion of the solute (CPA) as well as axial convection of the CPA, and a variable vascular surface area (A) during the transport process. In addition, the model also accounts for the radial movement of the solvent (water) into and out of the vascular spaces. Osmotic responses of various cells within an human ovarian tissue section are predicted by the numerical model with three model parameters: permeability of the tissue cell membrane to water (L(p)), permeability of the tissue cell membrane to the solute or CPA (omega) and the diffusion coefficient of the solute or CPA in the vascular space (D). By fitting the model results with published experimental data on solute/water concentrations within an human ovarian tissue section, I was able to determine the permeability parameters of ovarian tissue cells in the presence of 1.5M solutions of each of the following: dimethyl sulphoxide (DMSO), propylene glycol (PROH), ethylene glycol (EG), and glycerol (GLY), at two temperatures (4 degrees C and 27 degrees C). Modeling Approach 1: Assuming a constant value of solute diffusivity (D = 1.0 x 10(-9) m(2)/sec), the best fit values of L(p) ranged from 0.35 x 10(-14) to 1.43 x 10(-14) m(3)/N-sec while omega ranged from 2.57 x 10(-14) to 70.5 x 10(-14) mol/N-sec. Based on these values of L(p) and omega, the solute reflection coefficient, sigma defined as sigma = 1-omega v(CPA)/L(P) ranged from 0.9961 to 0.9996. Modeling Approach 2: The relative values of omega and sigma from our initial modeling suggest that the embedded ovarian tissue cells are relatively impermeable to all the CPAs investigated (or omega approximately 0 and sigma approximately 1.0). Consequently the model was modified and used to predict the values of L(p) and D assuming omega = 0 and sigma = 1.0. The best fit values of L(p) ranged from 0.44 x 10(-14) to 1.2 x 10(-14) m(3)/N-sec while D ranged from 0.85 x 10(-9) to 2.08 x 10(-9) m(2)/sec. Modeling Approach 3: Finally, the best fit values of D from modeling approach 2 were incorporated into model 1 to re-predict the values of L(p) and omega. It is hoped that the ovarian tissue cell parameters reported here will help to optimize chemical loading and unloading procedures for whole ovarian tissue sections and consequently, tissue cryopreservation procedures.     

Quantification of strains in biaxially tested soft tissues

A technique for the quantification of the strain field in the central region of biaxially tested planar soft tissues is presented. A vidicon-based image analysis system interfaced to a PDP 11/34 minicomputer is employed to track particles affixed to the specimen surface in real-time, from which the strains are inferred. Illustrative results are given for tests on excised canine pleural specimens on which four particles were affixed. The technique is applicable, however, to any planar soft tissue and any number of tracking particles. This procedure is recommended over previously used methods when testing anisotropic tissues.     

Fate of chloramphenicol in tobacco tissue culture under various light regimes

Abstract. Non-differentiated tissue cultures (calli) and differentiated tissues (shoots) of tobacco were found to differ in their sensitivity to chloramphenicol (CAP). This phenomenon is especially manifested in darkness and in an illumination regime lacking u.v. and blue light. When the latter are included, CAP's photodegradation products are shown to appear. It seems that one of the main photodegradation pathways is through the production of p -nitro-benzaldehyde ( p -NBA) which is further degraded. The possibility that either acetylation or physiological nitration of CAP is the cause for the differential tolerance was eliminated. The chromatographic and radiographic results indicate that in vivo degradation of CAP occurs both in calli and in shoot cultures. One of the in vivo degradation products is CAP-base.     

Sensitivity of random amplified polymorphic DNA analysis to detect genetic change in sugarcane during tissue culture

Random amplified polymorphic DNA (RAPD) analysis using 10-mer oligonucleotide primers efficiently differentiated sugarcane cultivars and proved suitable for detecting gross genetic change such as that which can occur in sugarcane subjected to prolonged tissue culture, for example in protoplast-derived callus. However, RAPD analysis was not sufficiently sensitive to detect smaller genetic changes that occur during sugarcane genetic transformation. The length of DNA scored for polymorphism per primer averaged 13.2 kb, or 0.0001% of the typical sugarcane genome size of 1.2 × 10₇ kb (2C). RAPD analysis of sugarcane plants regenerated from embryogenic callus revealed very few polymorphisms, indicating that gross genetic change is infrequent during this tissue culture procedure, although epigenetic effects result in transient morphological changes in regenerated plants. More sensitive variations on the RAPD technique may increase the practicality of DNA-based screening of regenerated plant lines to reveal somaclonal variants.