Multiweek cell culture project for use in upper-level biology laboratories.

This article describes a laboratory protocol for a multiweek project piloted in a new upper-level biology laboratory (BIO 426) using cell culture techniques. Human embryonic kidney-293 cells were used, and several culture media and supplements were identified for students to design their own experiments. Treatments included amino acids, EGF, caffeine, epinephrine, heavy metals, and FBS. Students researched primary literature to determine their experimental variables, made their own solutions, and treated their cells over a period of 2 wk. Before this, a sterile technique laboratory was developed to teach students how to work with the cells and minimize contamination. Students designed their experiments, mixed their solutions, seeded their cells, and treated them with their control and experimental media. Students had the choice of manipulating a number of variables, including incubation times, exposure to treatment media, and temperature. At the end of the experiment, students observed the effects of their treatment, harvested and dyed their cells, counted relative cell numbers in control and treatment flasks, and determined the ratio of living to dead cells using a hemocytometer. At the conclusion of the experiment, students presented their findings in a poster presentation. This laboratory can be expanded or adapted to include additional cell lines and treatments. The ability to design and implement their own experiments has been shown to increase student engagement in the biology-related laboratory activities as well as develop the critical thinking skills needed for independent research.

Transepithelial transport and metabolism of glycine in S1, S2, and S3 cell types of the rabbit proximal tubule.

In the first of two sets of experiments, the lumen-to-cell and cell-to-bath transport rates for glycine were measured in the isolated-perfused medullary pars recta (S3 cells) of the rabbit proximal tubule at multiple luminal glycine concentrations (0-2.0 mM). The lumen-to-cell transport of glycine was saturated, which permitted the calculation of the transport maximum of disappearance rate of glycine from the lumen (pmol.min(-1).mm tubular length(-1)), K(m) (mM), and paracellular leak (pmol.min(-1).mm tubular length(-1).mM(-1)) values for this transport mechanism; these values were 4.3, 0.3, and 0.03, respectively. The cell-to-bath transport did not saturate but showed a linear relationship to cellular glycine concentration, 0.58 pmol.min(-1).mm tubular length(-1).mM(-1). The second set of experiments characterized the transport rate, cellular accumulation, and metabolic rate of lumen-to-cell transported [(3)H]glycine in all segments (cell types) of the proximal tubule, pars convoluta (S1 cells), cortical pars recta (S2 cells), and medullary pars recta (S3 cells). These proximal tubular segments were isolated and perfused at a single glycine concentration of 11.2 microM. From the results of this study and previous work (Barfuss DW and Schafer JA. Am J Physiol 236: F149-F162, 1979), we conclude that the axial heterogeneity for glycine lumen-to-cell and cell-to-bath transport capacity extends to the medullary pars recta (S3 cells; S1 > S2 < S3 for lumen-to-cell transport and S1 > S2 > S3 for cell-to-bath transport). Also, we conclude that lumen-to-cell transported glycine can be metabolized and its metabolic rate displays axial heterogeneity (S1 > S2 > S3). The physiological significances of these transport and metabolic characteristics of the S3 cell type permits the medullary pars recta to effectively recover glycine from very low luminal glycine concentrations and makes glycine available for protective and maintenance metabolism of the medullary pars recta.

Luminal and basolateral membrane transport of glutathione in isolated perfused S(1), S(2), and S(3) segments of the rabbit proximal tubule.

Lumen-to-bath and bath-to-lumen transport rates of glutathione (GSH) were measured in isolated perfused S(1), S(2), and S(3) segments of the rabbit proximal tubule. In lumen-to-bath experiments, the perfusion solution contained 4.6 microM (3)H-GSH with or without 1.0 mM acivicin. In all three segments perfused without acivicin, luminal disappearance rate (J(DL)) and bath appearance rate (J(AB)) of (3)H-GSH were 14.5 +/- 0.5 and 2.2 +/- 0.8 fmol/min per mm tubule length, respectively. With acivicin present, J(DL) and J(AB) were reduced to 1.3 +/- 0.4 and 0.5 +/- 0.3, respectively, with no differences among segments. Cellular concentrations of (3)H-GSH in S(1), S(2), and S(3) segments when acivicin was absent were 23.1 +/- 2.0, 31.7 +/- 11.4, and 143.5 +/- 17.9 microM, respectively. With acivicin in perfusate, cellular concentrations were reduced but there was no change in the heterogeneity profile. In bath-to-lumen transport experiments (S(2) segments only), the bathing solution contained 2.3 microM (3)H-GSH. (3)H-GSH appearance in the lumen (J(AL), fmol/min per mm) and cellular accumulation from the bath were studied with and without acivicin in the perfusate. J(AL) values were 3.0 +/- 0.2 and 0.2 +/- 0.03 while cellular concentrations were 9.5 +/- 1.0 and 6.1 +/- 0.5 microM, respectively. It is concluded that: (1) GSH is primarily removed from the luminal fluid after degradation to glycine, cysteine, and glutamate, which are absorbed; (2) GSH can be absorbed intact at the luminal membrane; (3) the S(3) segment has the greatest GSH cellular concentration because its basolateral membrane has less capacity for cell-to-bath transport of GSH; and (4) GSH can be secreted intact from the peritubular compartment into the tubular lumen.