Primary kidney cells

Figure 10. Primary cultures of mouse kidney cells. Primary cultures of kidney epithelial cells derived from 10-day-old mice were grown either in hormonally defined medium with five supplements (5 pg/ml insulin, 5 pg/ml transferrin, 25 ng/ml PCE, 5 X10" M hydrocortisone, and 5 x 10" M Tj), or in medium supplemented with 10% fetal calf serum. After 10 days, primary cultures still were epithelial in morphology serum free (a) but were overgrown with fibroblasts with serum (b). (Taub et al., 1979 with permission.)...

A good example of the problems encountered with the use of serum with primary cultures is illustrated by the case with cultured kidney cells. The kidney epithelial cell line MDCK grows in serum-free medium supplemented with five supplements ... 

EPO in the human adult is synthesized almost exclusively by specialized kidney cells (peritubular interstitial cells of the kidney cortex and upper medulla). Minor quantities are also synthesized in the liver, which represents the primary EPO-producing organ of the foetus. 

Taub, M. (1984). Growth of primary and established kidney cell cultures in serum-free media. In Methods for Serum-Free Culture of Epithelial and Fibroblastic Cells (Barnes, D.W., Sirbasku, D.A.A. and Sato, G.H., Eds.). Alan R. Liss, New York, pp. 3-24. 

Weber E, Bannasch P Dose and time dependence of the cellular phenotype in rat hepatic preneoplasia and neoplasia induced by continuous oral exposure to N-nitrosomorpholine. Carcinogenesis 15 1235-42, 1994 Robbiano L, Mereto E, Corbu C, et ah DNA damage induced by seven N-nitroso compounds in primary cultures of human and rat kidney cells. Mutat Res 368(l) 41-7, 1996... 

The antiviral activity of 322, determined351 using primary rabbit kidney cells injected with HS V-l (Herpes Simplex Virus type 1), showed that BVFRU has antiviral potency and transport characteristics suitable for in vivo diagnosis of HSE (Herpes Simplex Encephal-ities) because of greater stability of bromine-carbon bonds than iodine-carbon bond present in [131I]IVDU352, [( )-5-(2-iodovinyl)-l-(2-deoxy- -D-ribofuranosyl)uracil]. 

As mentioned before, another relevant key development for the progress of animal cell culture technology was the WI-38 human diploid cell line obtained by Hayflick and Moorhead in 1961, since previously the options were the use of primary cultures or of heteroploid cell lines (derived from tumors or from cells that acquired tumor-like characteristics in culture). Because of that, heteroploid cells were not acceptable for the production of compounds for human applications, and therefore primary cultures from other species were employed (such as primary monkey kidney cells). 

A yield of about 108 cells/g of cortical tissue can be obtained and these should be diluted to 3 X 105 cell/ml and distributed into flasks for growth. Melnick s medium A (Melnick, 1955) was designed for growth of primary monkey kidney cells but other media have been devised for selective growth of cells of different origins ( 5.4). 

Macpherson (1961) exposed confluent monolayers or cell suspension to virus and then plated the cells at low density. They then picked out transformed colonies by appearance in the absence of any selective pressure (see Fig. 2.1). The number of transformed colonies is directly related to virus dose. When primary hamster kidney cells were exposed to polyoma virus at 96 p.f.u./cell between 1 in 100 and 1 in 5000 cells was transformed. 

The Chinese cell culture-derived inactivated vaccine is produced in primary hamster kidney cells a highly purified Vero cell culture-derived inactivated vaccine is under clinical development in France. A Chinese live-attenuated vaccine is also produced in primary hamster kidney cells. The efficacy of one dose is 80%, and the efficacy of two doses, given 1 year apart, is 97.5% this vaccine was evaluated in a randomized trial in 26 239 children, half of whom received the vaccine and half served as controls, and its adverse effects have been reviewed (SEDA-22, 350). 

Because NADPH-cytochrome P-450 reductase activity is highest in the cortex [77,121] and medullary microsomes lack cytochrome P450 [121], renal cortical tissue was used to investigate peroxidative injury caused by p-lactam accumulation in the kidney. Renal cortical microsomes, slices, tubule and cell suspensions, primary cultured renal cells and established kidney cell lines were exposed to -lactams with the aim to investigate the subcellular mechanism of the nephrotoxic injury. 

The purpose of this chapter is to present overviews of a selection of the major endothelial and epithelial barriers to drug delivery for which there are either primary culture or cell line systems that recapitulate the characteristics of the in vivo barrier. Our objective is to define some general characteristics of cell culture models and highlight the more commonly applied primary cell cultures and cell lines in use today. Specifically, we focus on cell culture models for the intestinal epithelium, blood-brain barrier, pulmonary and nasal epithelium, ocular epithelium, placental barrier, and renal epithelium. Renal epithelium was included here primarily because some cell lines derived from this tissue [e.g., Madin-Darby canine kidney cells (MDCK)] are often used as surrogates for other barriers by pharmaceutical scientists. We have arbitrarily chosen to exclude the skin and liver from the scope of this overview. However, it should be noted that hepatocyte cell culture models, for example, are becoming more widely available and have been the subject of recent reviews.1,2... 

The climate for permission by regulatory agencies, particularly by the FDA in the United States to use immortalized CHO cells for the production of recombinant proteins was not favorable in the early 1980s. Discussions about risks associated with the use of mammalian cells were controversial and had been initiated more than two decades earlier [134] when a first generation of classical biological products (i.e., vaccines and the natural interferons) were developed on the basis of primary monkey kidney cells, human diploid cells and, later, transformed mammalian cells. 

Growth substrate characteristics influence epithelial cell orientation and phenotypic expression due to the interaction of receptor sites on the cell surface with specific sites in the substrate/matrix. For example, growth of the Xenopus laevis kidney cell hne A6 on microporous substrates could induce the expression of vasopressin receptors absent in solid support grown monolayers [157]. Similar observations have been reported when rabbit primary cultures of renal cortical collecting ducts were maintained on microporous supports under constant medium perfusion [158]. 

Aflatoxin B leads to condensation of nuclei, separation of nuclei from the cytoplasm, cytoplasmic vacuolization, and loss of the brush border in MDBK (Madin-Darby bovine kidney) and PFBK cells (primary fetal bovine kidney) evidenced by electron microscopic examination [241]. In addition exposure of OK cells to Aflatoxin B resulted in an inhibition of inorganic phosphate uptake, which could not be abolished by application of parathyroid hormone (PTH) and insulin [221]. 

ANF has potent effects on cyclic GMP (cGMP) levels. In vitro, addition of atrial extracts to minced kidney tissue or to primary kidney cell cultures results in increased levels of cGMP.H Injection into anesthetized rats resulted in a 4-fold increase in plasma cGMP levels and a 28-fold increase in urinary excretion.Sodium nitroprusside also causes increased cGMP levels in treated tissues, but probably acts directly through stimulation of guanylate cyclase. Further studies must be done to clarify the role of cGMP in the vasorelaxant and the natriuretic actions of ANF. 

As summarized previously by this Committee, there was no evidence of DNA repair as a result of possible DNA damage in bacteria, whereas DNA single-strand breaks were consistently induced in cultured mammalian cells and were also observed in vivo in spleen, liver and kidney cells of mice after intraperitoneal injection of ochratoxin A. DNA repair, manifested as unscheduled DNA synthesis, was observed in most studies with primary cultures of rat and mouse hepatocytes, porcine epithelial cells from bladder and human urothelial cells (Annex 1, reference 153). 

In addition to major organs, such as a heart, lung, and liver, kidney- [30], splenon-[85, 86] and breast- [87] on-a-chip devices have been developed. Fig. 9 shows the schematics of these three devices. Kidney-on-a-chip devices include a porous membrane where kidney cells and epithelial are cultured in each side. This membrane, which is similar to the one used in the lung-on-a-chip device, consists of the central channel and two sub-channels—an apical luminal channel and a basal interstitial space. Compared to the traditional microfluidic system, the exposure of the epithelial cell layer to the certain shear stress generated by inflow mimics the in vivo kidney tubules, resulting in promotion of epithelial cell polarization and primary cilia formation. This platform is useful for the study of kidney toxicity during drug development. 

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