Enumeration of islets by nuclei counting and light microscopic analysis.

Islet enumeration in impure preparations by conventional dithizone staining and visual counting is inaccurate and operator dependent. We examined nuclei counting for measuring the total number of cells in islet preparations, and we combined it with morphological analysis by light microscopy (LM) for estimating the volume fraction of islets in impure preparations. Cells and islets were disrupted with lysis solution and shear, and accuracy of counting successively diluted nuclei suspensions was verified with (1) visual counting in a hemocytometer after staining with crystal violet, and automatic counting by (2) aperture electrical resistance measurement and (3) flow cytometer measurement after staining with 7-aminoactinomycin-D. DNA content averaged 6.5 and 6.9 pg of DNA per cell for rat and human islets, respectively, in agreement with literature estimates. With pure rat islet preparations, precision improved with increasing counts, and samples with about ≥160 islets provided a coefficient of variation of about 6%. Aliquots of human islet preparations were processed for LM analysis by stereological point counting. Total nuclei counts and islet volume fraction from LM analysis were combined to obtain the number of islet equivalents (IEs). Total number of IE by the standard method of dithizone staining/manual counting was overestimated by about 90% compared with LM/nuclei counting for 12 freshly isolated human islet research preparations. Nuclei counting combined with islet volume fraction measurements from LM is a novel method for achieving accurate islet enumeration.

Accurate control of oxygen level in cells during culture on silicone rubber membranes with application to stem cell differentiation.

Oxygen level in mammalian cell culture is often controlled by placing culture vessels in humidified incubators with a defined gas phase partial pressure of oxygen (pO(2gas)). Because the cells are consuming oxygen supplied by diffusion, a difference between pO(2gas) and that experienced by the cells (pO(2cell)) arises, which is maximal when cells are cultured in vessels with little or no oxygen permeability. Here, we demonstrate theoretically that highly oxygen-permeable silicone rubber membranes can be used to control pO(2cell) during culture of cells in monolayers and aggregates much more accurately and can achieve more rapid transient response following a disturbance than on polystyrene and fluorinated ethylene-propylene copolymer membranes. Cell attachment on silicone rubber was achieved by physical adsorption of fibronectin or Matrigel. We use these membranes for the differentiation of mouse embryonic stem cells to cardiomyocytes and compare the results with culture on polystyrene or on silicone rubber on top of polystyrene. The fraction of cells that are cardiomyocyte-like increases with decreasing pO(2) only when using oxygen-permeable silicone membrane-based dishs, which contract on silicone rubber but not polystyrene. The high permeability of silicone rubber results in pO(2cell) being equal to pO(2gas) at the tissue-membrane interface. This, together with geometric information from histological sections, facilitates development of a model from which the pO(2) distribution within the resulting aggregates is computed. Silicone rubber membranes have significant advantages over polystyrene in controlling pO(2cell), and these results suggest they are a valuable tool for investigating pO(2) effects in many applications, such as stem cell differentiation.

Effects of oxygen on mouse embryonic stem cell growth, phenotype retention, and cellular energetics.

Most embryonic stem (ES) cell research is performed with a gas phase oxygen partial pressure (pO(2)) of 142 mmHg, whereas embryonic cells in early development are exposed to pO(2) values of 0-30 mmHg. To understand effects of these differences, we studied murine ES (mES) growth, maintenance of stem cell phenotype, and cell energetics over a pO(2) range of 0-285 mmHg, in the presence or absence of differentiation-suppressing leukemia inhibitory factor (LIF). With LIF, growth rate was sensitive to pO(2) but constant with time, and expression of self-renewal transcription factors decreased at extremes of pO(2). Subtle morphological changes suggested some early differentiation, but cells retained the ability to differentiate into derivatives of all three germ layers at low pO(2). Without LIF, growth rate decreased with time, and self-renewal transcription factor mRNA decreased further. Gross morphological changes occurred, and overt differentiation occurred at all pO(2). These findings suggested that hypoxia in the presence of LIF promoted limited early differentiation. ES cells survived oxygen starvation with negligible cell death by increasing anaerobic metabolism within 48 h of anoxic exposure. Decreasing pO(2) to 36 mmHg or lower decreased oxygen consumption rate and increased lactate production rate. The fraction of ATP generated aerobically was 60% at or above 142 mmHg and decreased to 0% under anoxia, but the total ATP production rate remained nearly constant at all pO(2). In conclusion, undifferentiated ES cells adapt their energy metabolism to proliferate at all pO(2) between 0 and 285 mmHg. Oxygen has minimal effects on undifferentiated cell growth and phenotype, but may exert more substantial effects under differentiating conditions.