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Culture of skin in diffusion chamber. Culture of whole skin in diffusion chambers in autologous rabbits: maintenance of normal epidermal differentiation.

Pieces of thinly split whole skin were placed in diffusion chambers which were implanted intraperitoneally in autologous rabbits. The condition of pretreatment of the whole skin before the intraperitoneal implantation influenced greatly its viability at the initial stage of intraperitoneal culture: The whole skin incubated at room temperature for 1 hour in phosphate buffered saline containing 1 microgram/ml hydrocortisone could maintain almost completely the original structure of its epidermis showing complete keratinization successively as in vivo; without hydrocortisone in phosphate buffered saline, the culture resulted in degeneration of upper epidermis of the whole skin; meanwhile, the quick treatment of the whole skin with the buffer containing no hydrocortisone within 10 min resulted in partly slight parakeratosis. In the course of culture, new epidermis was formed under degenerated upper epidermis, showing complete keratinization. Epidermis could maintain nearly the original structure as long as 4 weeks of culture, however at 6 and 8 weeks of culture, it appeared flattened and partly degenerated. It was noticeable that in some part, the epidermal outgrowth formed directly on the filter showed complete keratinization.

Animals

Long-term survival of murine erythroid progenitors in long-term bone marrow culture and stromal cell culture: differentiation in peritoneal diffusion chamber culture.

Bone marrow cells of normal and cytosine-arabinoside (Ara-C) treated C57B1 mice were cultured in primary long-term culture (LTBMC) for a period of eight weeks. Non-adherent cells collected at weekly culture feedings consisted of neutrophils, macrophages and megakaryocytes. These were transferred into a) secondary peritoneal diffusion chamber cultures (DC) and b) secondary stromal cell cultures (SCC) first, and then into tertiary DC cultures. While in LTBMC and SCC there was no evidence of erythropoiesis, many erythroid colonies developed in DC cultures. It appears that undifferentiated erythroid progenitors may have a long survival in LTBMC and SCC devoid of erythropoietin and then differentiate in vivo in DC cultures in host mice without specific erythropoietic stimuli. Terminal differentiation and maturation of erythroid progenitors occurs to a limited extent in conventional DC cultures. The large number of erythroid colonies in DC observed in the present study could be due to increased sensitivity of undifferentiated erythroid progenitors from LTBMC to physiological levels of Epo in host mice of DC.

Animals

Osteogenesis in in vivo diffusion chamber cultures of human marrow cells.

The osteogenic diffusion chamber culture of rodent marrow cells is a well established system. In the present study, marrow cells from children and adult human donors were incubated in diffusion chambers implanted intraperitoneally in athymic mice. After 4 or 8 weeks, the chamber content was examined by light and electron microscopy. Child-cell cultures showed osteogenic tissue consisting of a mineralizing fibrous component and cartilage. Ultrastructurally, the fibrous tissue was similar to osteoid and exhibited osteoblast-like cells and mineralizing nodules. Mineral aggregates were also found in the cartilage. These features in child-cell chambers were similar to those found in control chambers of rabbit marrow cells. Adult-cell chambers showed only unmineralized fibrous tissue. These results render previous findings in animal-cell diffusion chamber systems relevant to the understanding of bone formation in man. It is suggested that the difference between child- and adult-cell chambers reflects an age-related decline in the number of marrow osteoprogenitor cells or their potential to undergo terminal osteogenic differentiation.

Adult

In vivo diffusion chamber culture reduces the tumorigenicity of PCC4-aza1 teratocarcinoma cells.

The established cells of strain 129 mouse teratocarcinoma PCC4-aza1 were cultured in diffusion chambers inplanted into the mouse peritoneal cavities. This unique culture reduced the tumorigenic activity of PCC4-aza1 cells without changing their F9 or H-2 antigenicity in a way similar to that described previously for embryoid body cells of OTT6050 teratocarcinoma cultured in diffusion chambers. These observations suggest that a reduction in the tumorigenicity of multipotent teratocarcinoma cells is generally not accompanied by a change in expression of F9 and H-2 antigenicity.

Animals

Comparative effects of sulfones and rifampin on growth of Mycobacterium lepraemurium in macrophage diffusion chamber cultures.

A cell-impermeable diffusion chamber technique has been developed that lends itself to growth studies of Mycobacterium lepraemurium. This technique, in which the organism grows within macrophage cultures inside the chambers that are maintained on monolayer cultures of macrophages, provides a method for a strict in vitro evaluation of antileprosy drugs without the influence of a multiplicity of host factors. This system was used to compare the effect of three sulfone derivatives and rifampin on the growth of M. lepraemurium within these diffusion chamber cultures. Two sulfones, 4,4'-diaminodiphenyl sulfone and 4,4'-diacetamidodiphenyl sulfone, as well as rifampin, suppressed the growth of M. lepraemurium, but monoacetyl sulfone 4-amino-4'acetamidodiphenyl sulfone had no effect. The results indicate that the diffusion chamber technique can be used to evaluate the inhibitory effect of antileprosy drugs on the growth of M. lepraemurium. Also, the method provides for the first time a relatively rapid in vitro method for directly comparing the effects of drugs or their analogs when outside the metabolic influence of an animal host. This technique may be a useful tool for chemotherapy studies with other antileprosy compounds.

Animals

[Capacity of erythroid progenitor cells of the human bone marrow for clonal growth in diffuse chamber culture].

The ability of human marrow erythroid progenitors growth in diffusion chamber culture has been assessed. Chambers containing cells (2 X 10(5)) suspended in clot were implanted into peritoneal cavity of anemic rats. The anaemia was induced by phenylohydrazine (50 mg/kg body weigh). Erythroid colonies were counted on day 1, 2, 3, 5, 7, 9, 11, 13 and 15. Two types of colonies formed by erythroid progenitors (BFU-D-E, CFU-D-E) were determined using criteria of colony size and culture duration. The count of colonies formed by CFU-D-E was highest on day 2 (29 +/- 4) and underwent reduction in subsequent days. Colonies formed by BFU-D-E were seen beginning of day 5 and their number peaked on day 7 and 9. These results indicate that the best time for determining the growth of human marrow CFU-D-E and BFU-D-E in diffusion chamber culture is on day 2 and day 7-9, respectively.

Animals

Fetal hemopoiesis in diffusion chamber cultures. I. The pattern of pluripotent stem cell growth.

The growth pattern of fetal liver (FL), normal adult bone marrow (NABM) and regenerating (post Velban treatment) adult bone marrow (RABM) colony forming units (CFU) cultured in diffusion chambers (DC) was studied. When twenty CFU were implanted into DC the recovery of CFU after 4 days with FL, NABM or RABM was 133 +/- 7, 19 +/- 2 and 34 +/- 2 CFU, respectively. The transplantation fraction of CFU from NABM decreased from 10-4% on day 0 to 6-9% on day 4; that of FL did not change from the initial 6-2%. The growth rate of CFU derived from FL was substantially greater than that from NABM. The relative growth of FL and RABM CFU was clearly inhibited when the concentration of cells cultured was increased. Spleen colonies from FL cells before culture were larger (P less than 0-005) than colonies from NABM but after 7 days of culture there was no difference between the two groups. Histological examination of spleen colonies showed that after DC culture FL and NABM CFU were differentiating along the three normal pathways. These data suggest that intrinsic differences exist between fetal and adult stem cells in the in vivo diffusion chamber culture system.

Animals

Growth and differentiation of rat bone marrow stem cells in diffusion chamber culture.

The growth of granulopoietic progenitors (CFU-C) and morphologically identifiable haemopoietic cells from rat bone marrow was studied in intraperitoneal diffusion chambers over a 6-day period. The aim of this study was to investigate whether stem cells preceding the granulopoietic progenitors are able to restore and to maintain depleted compartments of CFU-C and immature granulopoietic cells under the conditions of diffusion chamber culture. After treatment of rats with repeated doses of hydroxyurea (HU) a bone marrow cell suspension could be obtained wherein proliferating haemopoietic cells were reduced to a very low level and CFU-C to undetectable levels. During culture in diffusion chambers, growth of CFU-C, immature granulopoietic cells and macrophages could be observed from this HU-treated bone marrow. It is concluded that in diffusion chamber culture stem cells preceding CFU-C are able to rebuild functioning compartments of granulopoietic progenitors and morphologically identifiable haemopoietic cells.

Animals

Inhibition of osteogenesis by bone mass in diffusion chamber cultures.

The effect of increased bone mass on osteogenesis was studied using the diffusion chamber culture of mouse distal radius as an experimental model. Two distal thirds of a mouse's radii were cultured in separate diffusion chambers in a recipient mouse; one chamber contained only a distal radius, but the other contained a distal radius plus two proximal radii and two ulnae. The DNA synthesis of osteogenetic cells was significantly inhibited in cultures with excess bone mass. The mean inhibition in labelling indices of epiphyseal plate chondrocytes was 27% and of periosteal cells 40%. The nature of this inhibition seems to be specific.

Animals

Modulation of murine granulocyte proliferation in diffusion chamber cultures.

Normal mouse marrow cells were cultured in Millipore diffusion chambers for long periods of time and at a variety of cell concentrations. All cultures showed a pattern of granulocyte proliferation characterized by logarithmic growth followed by prolonged stabilization of cell number starting a 7 days thereafter. The height of this "plateau" varied in relationship to the level of cell input and characteristically was far lower than the maximum cell density that can be maintained in this culture system. Additional studies showed that the plateau represented a steady state of granulocyte turnover and was not due to alterations in the diffusion chambers or the host mice. Regulatory mechanisms intrinsic to the cultured cell population appeared to play a primary role in maintaining this stable plateau. Modulation of granulocyte proliferation was partly due to increasing cell density; particularly with high input concentrations. In addition, differential cell counts suggested that critical changes in the relationship between immature and mature granulocytes partially accounted for this apparent autoregulation of cell growth. The plateau period in diffusion chamber cultures in many ways resembles granulocyte proliferation in normal mouse bone marrow and is a useful model for the study of regulatory functions in granulocytopoiesis.

Animals

Ontogenesis of ultrastructural features during osteogenic differentiation in diffusion chamber cultures of marrow cells.

Three stages of osteogenic differentiation can be identified in in vivo diffusion chamber cultures (DCC) of unselected marrow cells, namely, proliferation, differentiation, and maturation (mineralization). These stages were characterized correlatively by in situ differential cell counts, alkaline phosphatase activity, and mineral accumulation. In the present study, the ultrastructure of marrow cell DCC was examined after incubation for 3-21 days. Features characteristic of osteoblastic and chondroblastic differentiation were first noted in 12 day DCC. Sites of osteoblastic differentiation showed cell-cell contacts associated with an increased cell density. The osteoblastic cells had long processes and were embedded in matrix with prominent fiber bundles reminiscent of collagen type I. The chondroblastic cells appeared solitary in areas of lesser cell density. By contrast to the long osteoblastic cell processes, they had short plasmalemmal projections and the matrix surrounding them contained single, thin, short fibers reminiscent of collagen type II, as well as proteoglycan granules. Both cell types showed prominent cytoskeletal elements, rough endoplasmic reticulum, and Golgi. One finding, previously unnoted in differentiating osteogenic cells, was mitochondria with condensed cristae that represent an increased rate of energy metabolism. These mitochondria were particularly abundant in the differentiation stage and declined as the cultures matured. These findings, together with previous reports in the epiphyseal growth plate, suggest that mineralization is associated with an optimal level of energy metabolism rather than extreme hypo- or hyperoxia. The set of ultrastructural parameters defined here in the marrow cell DCC may serve as useful markers for cells undergoing osteogenic differentiation.

Animals

Re-entry of resting leukaemic blood cells into proliferation in human acute leukaemia during diffusion chamber culture.

From 17 patients with different forms of acute leukaemia, mononuclear blood cells were cultured in diffusion chambers (DC) implanted intraperitoneally into pre-irradiated mice. In 14 patients, growth of blast cells could be observed during the culture period of up to 21 days. To question whether this growth of blast cells was due only to proliferation of the initially proliferating fraction or whether a re-entry of resting leukaemic cells into proliferation was involved, various 3H-thymidine (3H-TdR) labelling studies were carried out. The absolute increase of blast cells in EC showed no correlation with the fraction of leukaemic blast cells in DNA-synthesis in the implanted cell suspension as measured by 3H-TdR labelling in vitro. Furthermore, in 2 patients where the kinetic behaviour of initially labelled leukaemic blast cells was followed during DC culture, the increase in total blast cells could only be attributed to a small extent to proliferation of those cells initially in the cell cycle. Lastly, "in vivo" labelling during the culture period showed that in one case 25% and in another case 60% of the blast cells in DC were proliferating. The conclusion is that, owing to the stimulation of the diffusion chamber milieu and possibly also due to removal of an in vivo inhibition, in most cases of acute leukaemia resting leukaemic blast cells can apparently re-enter the active cell cycle. This has relevance for an understanding of the self-maintenance of the leukaemic cell population and may also be a reason for relapse of leukaemia after the usual cytostatic drug treatment which affects mainly the proliferating leukaemic cells.

Acute Disease

Microtopography of growth of normal and leukemic murine cells in diffusion-chamber cultures.

Histologic examination of "suspension" diffusion-chamber (DC) cultures of normal murine bone marrow cells demonstrates that hemopoietic cell growth in this system takes place in clonal form. Soon after implantation, marrow cells are arrayed on the filter membranes in a circumferential fashion adjacent to the surrounding lucite ring. Colonies of granulocytic cells soon form in this location and increase in size and number with increasing time of culture. Eventually cells begin to approach confluence over the entire filter membrane. Growth of C1498 murine acute myelogenous leukemic cells in DC cultures also takes place on the filter membranes and begins in a circumferential pattern. However, the leukemic cells grow diffusely and soon overspread the entire filter membrane, often growing several layers thick. Thus, although normal marrow cells are implanted into and are harvested from DC cultures in liquid-suspension form, they grow in a clonal pattern similar to that observed with the plasma-clot and fibrin-clot DC culture methods.

Animals

Macrophages and/or fibroblasts in hematopoietic diffusion chamber cultures.

An ultrastructural study of diffusion chamber (DC) hematopoietic colonies derived from rat bone marrow is reported. The emphasis is placed on the structural features of macrophages and fibroblasts in cultures. The observations indicate that the two cell types are normal components of DC cultures. In older cultures both macrophages and fibroblasts contain large lipid droplets, sometimes causing difficulty in the precise characterization of the cell type. The possible origin of fibroblasts from adventitial cells if sinuses or of larger blood vessels of the bone marrow implanted in diffusion chambers is discussed. It is suggested that fibroblasts exert an enhancing influence on the growth of blood cells in DC cultures, comparable to feeder layers in in vitro cultures.

Animals

Evidence for differentiation of human leukemic blood cells in diffusion chamber culture.

Peripheral blood cells of 21 patients with different forms of acute leukemia were cultured in diffusion chambers (5 x 10(5) cells/chamber) implanted intraperitoneally in 650 R preirradiated host mice over a period of up to 21 days. In patients with acute myeloid leukemia (AML), acute erythroleukemia (AEL), or acute myelomonocytic leukemia (AMMoL), the total number of cells which developed during this culture period exceeded the implanted value and also the values for normal peripheral blood cells from ten controls. In acute undifferentiated leukemia (AUL), two out of six patients showed considerable growth whereas the others, and also two patients with acute lymphoid leukemia (ALL), had poor growth. Differential counts revealed that the rise in total cells was due mainly to proliferation of blast cells and formation of granulopoietic cells. The latter exceeded the numbers from normal peripheral blood cells in 9 out of 13 patients with AML, AEL, or AMMoL and in 2 out of 6 patients with ALL. The production of granulopoiesis was not restricted to proliferating cells, but included mature cells which were of abnormal morphology in some cases. From the amount of granulopoiesis and the time of its development it was assumed that they were at least partly derived from leukemic blast cells. Chromosome analyses to decide whether the granulopoietic cells were of leukemic or normal cell origin are in progress.

Acute Disease

Diffusion chamber cultures of muscle from patients with scoliosis.

Explants from paraspinal muscles of patients with scoliosis were cultured in Millipore diffusion chambers placed in peritoneal cavities of mice. Three and four weeks of incubation produced a predominantly myogenic growth. Cell counts done on paraffin sections and on dissociated cells from the chambers showed that explants from the concavity of the spinal curvature grew better than those from the convexity. Since cells from injured muscles, when transplanted or cultured, are known to grow better than those from healthy control tissue, these findings support our previous morphologic observations that in idiopathic scoliosis concave muscles are more involved by the disease process. Secondary subculture of the chamber contents by conventional methods produced myotubes within one to two weeks. The relative ease of diffusion chamber muscle culture should make the method useful to laboratories that are not specifically equipped for tissue culture work.

Adolescent

Leukemic host influence on normal erythrocytic and granulocytic colony formation in in vivo plasma clot diffusion chamber cultures.

The effect of a leukemic environment on normal erythroid and granulocytic colony formation was examined in in vivo plasma clot diffusion chamber cultures implanted into Shay chloroleukemic rat hosts at varying stages of the disease. Normal bone marrow cells isolated in plasma clot diffusion chamber cultures in leukemic hosts displayed significant differences in the pattern of normal bone marrow colony growth. Granulocyte colony-forming units were significantly inhibited by leukemic hosts throughout the course of the disease. The size of developing colonies was reduced to under 100 cells; however, maturation within these clusters appeared unaffected. Erythroid colonies showed a slight inhibition during the early stages of the leukemia, a significant stimulation of 100 to 350% in the midleukemic period, and a significant inhibition of 50 to 65% during the terminal stages of the disease. Burst formation was also inhibited in the late leukemic stages. The transient increase in erythroid colony-forming units on Days 7 and 8 of the leukemia was concomitant with the onset of the anemia associated with the disease. Since the normal bone marrow cells were compartmentalized within the plasma clot diffusion chamber cultures, the suppression of erythroid and granulocytic colony development appears to be directly due to the release of diffusible inhibitory substances from the leukemic animal.

Animals

Cell growth and differentiation of murine extra-embryonic fetal and adult hematopoietic tissues in diffusion chamber cultures.

Studies were conducted with diffusion chambers (DC) filled with cell suspensions from different CF1 murine hematopoietic tissues: adult peripheral blood; adult tibial marrow; day 17-5 of gestation fetal liver, spleen and thymus; day 14-5 gestation fetal liver; day 10-5 of gestation yolk sac. After an initial decrease in DC cell numbers on day 2 of culture, growth of each cell group continued, but, at different rates. ATM had the highest growth ratio and FT-D17-5(2) had the lowest. The growth rates for APB and FL-D17-5 were similar. FS-D17-5 and FL-D14-5 cultures did not recover from the day 2 values (i.e. FL-D14-5 DC values on day 13-14 of culture were half that recorded on day 2). The YS-D10-5 DC cell numbers continued to increase throughout the 14 days of study. The profile of cellular elements from the DCs did not reflect the original cell suspensions. The predominant cell type recovered from peripheral blood cultured for 14 days was the macrophage. By day 10-14 of culture, the populations of cells harvested from the fetal tissue DC groups were similar to that of tibial marrow. Both proliferative and mature granulocytes, and macrophages were the predominant cell types. The yolk-sac pattern of cytodifferentiation recorded on day 7-14 was unlike that of the other groups. These DC cultures were comprised of mainly macrophages and plasma cells.

Animals