Successful scale-up of human embryonic stem cell production in a stirred microcarrier culture system

Future clinical applications of human embryonic stem (hES) cells will require high-yield culture protocols. Currently, hES cells are mainly cultured in static tissue plates, which offer a limited surface and require repeated sub-culturing. Here we describe a stirred system with commercial dextran-based microcarriers coated with denatured collagen to scale-up hES cell production. Maintenance of pluripotency in the microcarrier-based stirred system was shown by immunocytochemical and flow cytometry analyses for pluripotency-associated markers. The formation of cavitated embryoid bodies expressing markers of endoderm, ectoderm and mesoderm was further evidence of maintenance of differentiation capability. Cell yield per volume of medium spent was more than 2-fold higher than in static plates, resulting in a significant decrease in cultivation costs. A total of 10(8) karyotypically stable hES cells were obtained from a unitary small vessel that needed virtually no manipulation during cell proliferation, decreasing risks of contamination. Spinner flasks are available up to working volumes in the range of several liters. If desired, samples from the homogenous suspension can be withdrawn to allow process validation needed in the last expansion steps prior to transplantation. Especially when thinking about clinical trials involving from dozens to hundreds of patients, the use of a small number of larger spinners instead of hundreds of plates or flasks will be beneficial. To our knowledge, this is the first description of successful scale-up of feeder- and Matrigel™-free production of undifferentiated hES cells under continuous agitation, which makes this system a promising alternative for both therapy and research needs.

Death in dish: controls of apoptosis within the developing retinal tissue

Studies of programmed cell death in the developing retina in vitro are currently reviewed. The results of inhibiting protein synthesis in retinal explants indicate two mechanisms of apoptosis. One mechanism depends on the synthesis of positive modulators ('killer proteins'), while a distinct, latent mechanism appears to be continuously blocked by negative modulators. Extracellular modulators of apoptosis include the neurotrophic factors NT-4 and BDNF, while glutamate may have either a positive or a negative modulatory action on apoptosis. Several protein kinases selectively modulate apoptosis in distinct retinal layers. Calcium and nitric oxide were also shown to affect apoptosis in the developing retianl tissue. The protein c-Jun was found associated with apoptosis in various circumstances, while p53 seems to be selectively expressed in some instances of apoptosis. The results indicate that the sensitivity of each retinal cell to apoptosis is controlled by multiple, interactive, cell type- and context-specific mechanisms. Apoptosis in the retina depends on a critical interplay of extracellular signals delivered through neurotrophic factors, neurotransmitters and neuromodulators, several signal transduction pathways, and the expression of a variety of genes.

Apoptosis in the developing retina: paradoxical effects of protein synthesis inhibition

Cell death by apoptosis is usually characterized as an active process that requires protein and RNA synthesis. The requirement of protein synthesis for the degeneration of ganglion cells and other cell types was studied in neural retinae explanted from the eyes of newborn rats. Ganglion cells were detected by the presence of retrogradely transported horseradish peroxidase injected into the superior colliculus. Apoptotic cells were recognized by their condensed and deeply stained chromatin. The data show that the death of ganglion cells, whose axons are damaged when preparing the explants, is blocked or delayed by protein synthesis inhibitors. In contrast, the blockade of protein synthesis produced cell death with apoptotic morphology in the neuroblastic layer of the same retinae. The results suggest the operation in the developing retina of both a program of apoptosis dependent on the synthesis of killer proteins, and a latent mechanism of apoptosis that is normally blocked by repressor proteins.

Intraretinal neurotrophic activity prevents the degeneration of ganglion cells in retinal explants

The degeneration of ganglion cells was studied in neural retina explanted from the eyes of newborn rats. The ganglion cells were detected by the presence of retrogradely transported horseradish peroxidase injected into the superior colliculus. The time course of cell death among the axotomized ganglion cells in the explants was similar to that found in vivo after axotomy in neonatal rats. The effect of culture media conditioned with retinal cells from either newborn rats or chick embryos was tested on the survival of ganglion cells in the explants. Both conditioned media increased 2- to 3-fold the survival of rat retinal ganglion cells after 2 days in culture. The data show that soluble trophic factors released by retinae of distinct species can influence the survival of ganglion cells within their histotypic microenvironment

Evidence for a developmentally regulated neurotrophic activity from chick retina affecting the survival of retinal ganglion cells from newborn rats

The effect of conditioned medium from aggregates of chick embryo retinal cells was tested on the in vitro survival of retinal ganglion cells were identified by the detection of retrogradely transported horseradish peroxidase injected bilateral into the superior colliculus. Culture medium conditioned with chick embryo retinae was tested on monolayers of rat retinal cells with plating densities ranging from 0.5 to 4 x 10 5 cells/cm2. In all cases the conditioned medium significantly increased the survival of ganglion cells after 2 days in culture. Conditioned media from embryonic days 8 to 16 (E8 to E16) presented neurotrophic activity, with the greatest effect occurring at E10-E12. The conditioned medium had no effect on the adhesion of rat retinal cells. The data suggest that chick retinal cells produce soluble trophic factors which can influence the survivalof rat retinal ganglion cells in vitro. Furthermore, the release of this neurotrophic activity by chick retina seems to be developmentally regulated