Demonstrating ß-glucan and yeast peptide clearance in biopharmaceutical downstream processes.

The use of yeast- and plant-derived hydrolysates in cell culture production processes has sparked concerns over the potential immunogenicity risk posed by ß-glucans and yeast peptides contained in these raw materials. This article utilizes a combination of in-process testing from large-scale manufacturing and scale-down spiking studies to demonstrate the clearance of ß-glucans and yeast peptides through chromatographic steps in the downstream purification process for a monoclonal antibody. ß-Glucans were found to flow through most all three modes of chromatography (Protein A, cation and anion exchange) without binding to the resins or the product. Protein A affinity chromatography was found to provide the best clearance factor. The efficacy of the resin sanitization and storage procedures to prevent carryover from one run to the next was also demonstrated. Yeast peptides were found to be metabolized during the cell culture process and were undetectable after the Protein A purification step. The data presented here serve to allay concerns about the use of hydrolysates in cell culture production. The methodology presented here provides a template to demonstrate clearance of ß-glucans and yeast peptides through chromatographic steps in downstream processing.

Comparison of aerobic standard medium with specific fungal medium for detecting fusarium spp in blood cultures.

In order to compare the performances of a specific fungal medium and a standard aerobic medium for detecting growth of Fusarium spp. in blood, simulated blood cultures were performed. For lower inocula (10(2) and 10(3) cfu/ml taken together), fungal growth was detected significantly earlier using the fungal medium. The mean difference in the time to detection between the two media was 22.33 h at 10(2) cfu/ml, with the maximum difference being achieved for Fusarium verticilloides at 37.05 h. These in vitro test results suggest fungal medium could be useful for obtaining more rapid blood culture results when evaluating patients at risk for invasive infection with Fusarium spp.

[Effect of the supernatant from mice liver cell primary culture on the in vitro growth of Plasmodium falciparum wild isolates]. / Effet du surnageant de culture primaire d'hépatocytes de souris sur la prolifération in vitro des isolats sauvages de Plasmodium falciparum.

Cultivation of Plasmodium falciparum has been a major research success, leading to a greater understanding of the parasite. Despite the fact that several P. falciparum clones have been maintained in continuous culture in different laboratories, research in genomics and proteomics would require parasitic material produced from fresh wild isolates. We have tested the effect of the supernatant from primary culture of mice hepatocytes on in vitro growth of P. falciparum isolates. Parasitized blood samples were collected from Madagascan malarious patients naturally infected. Isolates proliferation was assessed by use of isotopic method. The asexual erythrocytic stages of P. falciparum were grown for 42 hours in RPMI 1640-based medium plus L15 medium-based supernatant from mice liver cells culture, and in standard RPMI 1640-based medium alone. The mean of parasite growth was 1.5 times greater when the standard medium was enriched with the liver cells layer supernatant at a proportion of 10% and 15% (v/v). The usefulness of P. falciparum ex-vivo culture and of the hepatocytes in vitro primary culture is discussed.

Long-term culture of murine epidermal keratinocytes.

The production of transgenic and null mice with skin abnormalities makes it increasingly important to establish cultures of mouse epidermal keratinocytes for in vitro studies. This requires that each cell line be derived from a single mouse and that the cells be carried for multiple passages. Freezing the cells would also be advantageous by allowing comparison of keratinocytes from several mouse lines at the same time. Mouse keratinocytes, however, have been exceedingly difficult to grow as primary cultures, and subculturing these cells has been virtually impossible until now. We describe a gentle dissociation method and a highly supplemented fibroblast conditioned medium that allows us to grow and subculture total mouse keratinocytes for up to 19 subcultures, allowing an increase in cell number of greater than 10 logs. Epidermal keratinocytes from newborn mice were grown on collagen IV coated dishes in murine fibroblast conditioned medium with 0.06 mM calcium and added growth factors. The cells could be passaged, frozen as viable stocks, and induced to differentiate. Morphologically the cultured keratinocytes demonstrated a pattern characteristic of basal cells. Stratified cultures which made mouse keratin 1 and profilaggrin through passage 10 were induced by purging the monolayer cultures of growth factors, then adding medium with 0.15 mM calcium; expression of mouse keratin 1 and profilaggrin was lost by passage 15. The methods explained in detail here should be of great interest to investigators who are now trying to analyze skin phenotypes and expression of markers of epidermal differentiation of their transgenic or knockout mice.

[Preparation and testing of Sardinella peptones: application to lipase production by Staphylococcus sp]. / Elaboration de peptones de Sardinelle: application à la production de lipase par la souche Staphylococcus sp.

Production of lipase by Staphylococcus sp. in media containing fish peptones from sardinelle (Sardinella aurita) prepared in the laboratory was studied. Lipase production is strongly affected by lipids present in fish flours. Fish peptones prepared from dIgresed whole flesh was an excellent substrate for lipase production. A comparison of lipase production in media containing fish peptones or high quality commercial peptones indicated that fish peptones enhanced enzyme formation.

A sensitive method for testing the quality of organ culture media and of individual medium components in a cornea bank.

BACKGROUND: It has been suggested that variations in the quality of organ culture preservation media are responsible for variations in early postoperative graft morphology. Spates of such variations have been observed repeatedly for short periods. This paper reports the results of a series of grafts with low postoperative clearing observed during a period of 6 weeks. Simultaneously, preoperative phase-contrast microscopy evaluation of the corneal endothelium revealed that an unusually large proportion of donor corneae were unsuitable for transplantation. METHODS: The corneal storage media were therefore rigorously screened, paying particular attention to specific components and properties of the medium, including L-glutamine, amphotericin B, water quality, pH, and the glassware used. Possible toxic effects were identified by means of a sensitive growth assay performed using isolated human corneal endothelial cells. RESULTS: The evaluation demonstrated that both the water quality and the L-glutamine which had been used for preparation of the medium were substandard during the period in which poor clinical results were obtained. CONCLUSION: It is recommended that cornea banks undertaking long-term organ culture use standardized protocols and carefully monitored equipment. The quality of the basal media and supplements should be routinely checked.

An evaluation of media for transport of tissues infected with Borrelia burgdorferi.

Currently, the best medium for culture of Borrelia burgdorferi, the etiologic agent of Lyme disease, is Barbour-Stoenner-Kelly (BSK), or its modifications. This medium is complex, expensive, and laborious to prepare. A recent report suggested that a less expensive and simpler medium, hypertonic Columbia broth, might be useful as a transport medium for human tissues infected with B burgdorferi. To test this observation, hypertonic Columbia broth, Amies broth, distilled water, physiologic saline, phosphate-buffered saline (PBS), and modified Stuart medium were compared with BSK II as transport media, using ear and tail tissue samples from B burgdorferi-infected laboratory mice and using holding times and temperatures simulating actual transport conditions. The results showed BSK II to be markedly superior to the other media tested, although B burgdorferi remained viable in a few tissue samples held at room temperature in hypertonic Columbia broth, physiologic saline, or PBS for up to 2 days. Barbour-Stoenner-Kelly II continues to be the best medium for transport of tissues infected with B burgdorferi.

The effect of coculture on human zygote development.

It is well over a decade since the birth of the first test-tube baby and still the in-vitro conditions for early embryonic development remain suboptimal. The ideal culture medium to increase longevity and improve viability of human embryos is not available. Since the metabolic requirements of the human embryo changes from one cleavage stage to another, the development of a single culture medium for all stages could not be expected. The use of helper cells (coculture) in vitro offers much promise as there are numerous documentations in both man and animals describing their ability to increase blastulation rates and improve embryo viability. This paper reviews the effect of coculture on human zygote development. The selection and establishment of cell-lines, biologic actions of coculture of gametes and zygotes, the outcome, and future prospects are discussed.