Controlled environment culture of bone marrow explants from human myeloma.

Bone marrow biopsy specimens from patients with myeloma were cultured in either 1 of 2 thin-film culture systems, a controlled environment steady state system or a rocker tube configuration of the system, for periods up to 42 days. Both functional and morphological characteristics of the myeloma cells were well-maintained in these systems. Cytocentrifuge preparations of the culture media disclosed hematopoietic cells that included from 5% to almost 100% plasma cells. Histological examination of the cultured specimens disclosed infiltration of the marrow with myeloma cells. Myeloma proteins were released at a steady rate throughout the period of culture after the 1st 4 days. Bone-resorbing activity was demonstrated in the culture media in 7 of 9 myeloma culture media and was well maintained, particularly during the 1st week of culture. This activity was associated with severe osteolytic lesions in the donor patient and marked infiltration of the cultured specimen by myeloma cells. The potential use of these organ culture systems for the further definitive identification of the factor responsible for bone destruction in myeloma is discussed.

Biochemical actions of fibroblast kinin-forming protease.

Purification and further characterization of a kinin-forming acid protease in a mouse fibroblast L929 stationary cell culture line was carried out. Supernatants of dialyzed fibroblast homogenates digested denatured hemoglobin at pH 4.0. The supernatant was fractionated on a G-200 Sephadex column, hydroxylapatite column and finally on a DEAE-A50 Sephadex ion exchange column. A 9.4 fold purification was achieved with a 13.8% yield. The enzyme had a specific activity of 2062 ng kinin per mg protein when measured on a purified rat kininogen using the isolated rat uterus as the bioassay tissue. The protease had a pH optimum of 3.8-4.0. Molecular weights of the enzyme and substrate estimated on a G-200 Sephadex column were 39,000 and 110,000 respectively. Kinin formation was a function of both incubation time and enzyme concentration. Protease activity was localized primarily in the 10,000 g supernatant cell fraction (61.5%) with the 1500 g precipitate cell fraction containing 38.5% of the activity.

Ammonia inhibition of interferon synthesis.

Ammonium chloride (NH4Cl) was found to markedly inhibit the ability of cultured human fibroblasts to establish an antiviral state following exposure to poly IC. This antiviral state was diminished by the simultaneous addition of as little as 200 microgram/ml of NH4Cl. The effects of ammonia on the superinduction of human fibroblast interferon (IFN-beta) were also investigated. The titer of IFN dropped from 2600 units/ml in control cultures, to less than 50 units/ml in the presence of 400 microgram/ml of NH4Cl. A critical stage sensitive to ammonia was within the first 15 minutes following addition of poly IC.

Controlled culture of adult human skin: characterization.

Long term growth and differentiation of adult human skin cells has been achieved by more effective control of culture pH, gas phase, osmolarity and nutrient supply at optimized set point values than that possible with conventional batch feed culture. This control was achieved by means of an automatic perfusion, rocker culture apparatus. The following characteristics of these skin cultures particularly lend themselves to application as a source of skin graft material for use in humans requiring large area skin grafts: high culture success rate was achieved in that 12/12 specimens of adult skin exhibited growth to confluence within 4-6 weeks. Cultures demonstrated logarithmic growth for a minimum of 5 weeks, 25-50 fold enhancement in area by 4-6 weeks, 16-fold increase in glucose utilization at 9 weeks, nearly 4-fold increase in total DNA at 5 weeks, and a minimum 6-fold increase in nuclei counts at 5 weeks in culture. The multilayered outgrowth maintained a predominantly epithelial cell composition, but contained normal skin cell types other than keratinocytes, including melanocytes, and dermal fibroblasts, which may be required for long term survival of cultured skin transplanted back to humans. Other than the use of 10% fetal bovine serum in culture medium and denatured bovine collagen substratum, the system did not require heterologous components for plating or growth stimulation. Neither mouse feeder cells, nor the selective pressures of passage were required to obtain growth in surgically useful quantities.(ABSTRACT TRUNCATED AT 250 WORDS)

Steady-state cultures of human skin.

Pretransplantation cultivation of adult human skin has been optimized for rapid and prolonged outgrowth of epidermal cells from tissue explants using autonomic-perfusion, thin-layer culture technology (steady-state). This system fostered growth of autologous mesenchymal elements via critical control of the culture environment. The resulting cellular outgrowth maintained a balanced epithelial-dermal relationship, contained keratinocytes as well as minority epidermal cells, melanocytes and possibly Langerhans cells. Critical control of culture pH and osmolarity was found to enhance epithelial cell proliferation.

Culture human skin: heterotransplantation.

We have reported that human skin, cultured in a controlled environment system (steady state), yields epidermis-like growth from adult donor split thickness specimens. A procedure for transplantation of such cellular outgrowths to athymic nude mice for functional and morphological evaluations is reported. Successful transplants were definitely scored by the presence of histologic epidermal markers and human glucose phosphate isomerase. We suggest that the transplantation procedures reported here may contribute to the knowledge required for the use of autologous cultured skin in patients with large skin wounds.

Studies on normal human bone marrow cultures in controlled environment thin-film culture systems.

Two thin film culture systems, the controlled environment steady state system (SS) and the rocker tube configuration of that system (RT), were used to identify some of the conditions that appear to maintain morphologic and functional characteristics of cells of human bone marrow explants in vitro. The systems configuration assured continual gassing, control and easy monitoring of the cultures. Cytocentrifuge preparations of media of specimens cultured in RT disclosed, though in decreasing numbers, various hematopoietic cells for periods exceeding one month. Hematopoietic cells shed from specimens cultured in the SS system were retained in the culture tubes; cells of the myelocytic series predominated for the first two weeks while an increasing number of monocytes and macrophages appeared in the media of of older cultures. Histologic examination of cultured explants disclosed preservation of the marrow architecture and the persistence of hematopoietic cells. Specimens cultured in RT tubes tended to be less cellular than similar cultures placed in dialysis bags or as cultured in the SS system. Immunoglobulins (Ig) were released into the culture media at a constant rate throughout the period of culture. Specimens that were cultured at a controlled pH of 7.4 released 2 to more than 4 times as much Ig as similar specimens maintained at a pH level of 7.1. There were no definitive differences in Ig levels in the cultures maintained at comparable pH levels and overlaid with various CO2 concentrations, i.e. 2%, 5%, 10% similarly, no differences in Ig levels were found in specimens cultured in media containing fetal bovine sera as opposed to horse sera.

Culture of mammalian prostatic cells.

Preliminary experimental data and concepts oriented toward the achievement of certified primary prostatic cell cultures, normal and/or malignant, for mass distribution are presented. Such monolayer cultures would be established as subcultures from the microcarrier, spin-filter (steady-state) system. The suspension culture of primary cells directly derived from the tissue of origin would be propagated at high cell populations for extended periods of time. The ease of complete monitoring on a sequential basis for conformity to specifications is apparent. The feasibility of the approach appears to have been established. Problems relating to the reduction to practice are detailed.