Installing oncofertility programs for common cancers in limited resource settings (Repro-Can-OPEN Study): An extrapolation during the global crisis of Coronavirus (COVID-19) pandemic.

PURPOSE: The state of limited resource settings that Coronavirus (COVID-19) pandemic has created globally should be taken seriously into account especially in healthcare sector. In oncofertility, patients should receive their fertility preservation treatments urgently even in limited resource settings before initiation of anticancer therapy. Therefore, it is very crucial to learn more about oncofertility practice in limited resource settings such as in developing countries that suffer often from shortage of healthcare services provided to young patients with cancer. METHODS: As an extrapolation during the global crisis of COVID-19 pandemic, we surveyed oncofertility centers from 14 developing countries (Egypt, Tunisia, Brazil, Peru, Panama, Mexico, Colombia, Guatemala, Argentina, Chile, Nigeria, South Africa, Saudi Arabia, and India). Survey questionnaire included questions on the availability and degree of utilization of fertility preservation options in case of childhood cancer, breast cancer, and blood cancer. RESULTS: All surveyed centers responded to all questions. Responses and their calculated oncofertility scores showed different domestic standards for oncofertility practice in case of childhood cancer, breast cancer, and blood cancer in the developing countries under limited resource settings. CONCLUSIONS: Medical practice in limited resource settings has become a critical topic especially after the global crisis of COVID-19 pandemic. Understanding the resources necessary to provide oncofertility treatments is important until the current COVID-19 pandemic resolves. Lessons learned will be valuable to future potential worldwide disruptions due to infectious diseases or other global crises.

Phase II study of interim PET-CT-guided response-adapted therapy in advanced Hodgkin's lymphoma.

BACKGROUND: Combination chemotherapy ABVD (doxorubicin, bleomycin, vinblastine and dacarabazine) cures ∼70% of patients with advanced Hodgkin's lymphoma (aHL, stages IIB, III and IV) while more toxic escalated BEACOPP (EB, combination of bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine and prednisolone) increases cure rates to 85%. Patients with a positive interim positron emission tomography-computerized tomography (PET-CT) scan after two cycles (PET-2) of ABVD have very poor outcomes with continued ABVD. Intensifying therapy with EB in PET-2-positive patients ('response-adapted therapy') may improve cure rates, whereas the negative patients can continue ABVD alone. PATIENTS AND METHODS: Eligible patients with newly diagnosed aHL received two cycles of ABVD and underwent PET-2 (scored with semi-quantitative 5-point visual criteria, 'Deauville score'). PET-2-negative patients continued four additional cycles of ABVD, whereas PET-2-positive patients received four cycles of EB. A phase II sample size of 50 was estimated keeping the lower and higher proportion of rejection of the event-free survival (EFS) as 70% and 85%, respectively. RESULTS: Fifty patients [median age 28 (12-60) years; male : female: 39 : 11; stages: IIB-3 (6%), III-29 (58%) and IV-18 (36%); International Prognostic Score (IPS): 0-3: 34 (68%); 4-7: 16 (32%)] were enrolled; 49 underwent PET-2. Eight (16%) were PET-2-positive, whereas 41 (84%) were negative. Forty-seven were evaluable for EFS and all 50 for overall survival (OS). The 2-year EFS was 76% (95% CI: 68-83) and OS was 88% (95% CI: 82-94). PET-2 was strongly prognostic-2-year EFS, negative versus positive: 82% versus 50%; P = 0.013. CONCLUSION: PET-2 response-adapted strategy could not achieve EFS of 85% in aHL. However, escalated therapy improved outcomes in PET-2-positive patients compared with historical data. TRIAL REGISTRATION: CTRI/2012/06/002741 (http://www.ctri.nic.in) and NCT01304849 (http://www.clinicaltrials.gov).

Influence of BDNF Val66Met polymorphism on excitatory-inhibitory balance and plasticity in human motor cortex.

OBJECTIVE: While previous studies showed that the single nucleotide polymorphism (Val66Met) of brain-derived neurotrophic factor (BDNF) can impact neuroplasticity, the influence of BDNF genotype on cortical circuitry and relationship to neuroplasticity remain relatively unexplored in human. METHODS: Using individualised transcranial magnetic stimulation (TMS) parameters, we explored the influence of the BDNF Val66Met polymorphism on excitatory and inhibitory neural circuitry, its relation to I-wave TMS (ITMS) plasticity and effect on the excitatory/inhibitory (E/I) balance in 18 healthy individuals. RESULTS: Excitatory and inhibitory indexes of neurotransmission were reduced in Met allele carriers. An E/I balance was evident, which was influenced by BDNF with higher E/I ratios in Val/Val homozygotes. Both long-term potentiation (LTP-) and depression (LTD-) like ITMS plasticity were greater in Val/Val homozygotes. LTP- but not LTD-like effects were restored in Met allele carriers by increasing stimulus intensity to compensate for reduced excitatory transmission. CONCLUSIONS: The influence of BDNF genotype may extend beyond neuroplasticity to neurotransmission. The E/I balance was evident in human motor cortex, modulated by BDNF and measurable using TMS. Given the limited sample, these preliminary findings warrant further investigation. SIGNIFICANCE: These novel findings suggest a broader role of BDNF genotype on neurocircuitry in human motor cortex.

Practical Consensus Recommendations for Optimizing Risk versus Benefit of Chemotherapy in Patients with HR Positive Her2 Negative Early Breast Cancer in India.

Breast cancer is a public health challenge globally as well as in India. Improving outcome and cure requires appropriate biomarker testing to assign risk and plan treatment. Because it is documented that significant ethnic and geographical variations in biological and genetic features exist worldwide, such biomarkers need to be validated and approved by authorities in the region where these are intended to be used. The use of western guidelines, appropriate for the Caucasian population, can lead to inappropriate overtreatment or undertreatment in Asia and India. A virtual meeting of domain experts discussed the published literature, real-world practical experience, and results of opinion poll involving 185 oncologists treating breast cancer across 58 cities of India. They arrived at a practical consensus recommendation statement to guide community oncologists in the management of hormone positive (HR-positive) Her2-negative early breast cancer (EBC). India has a majority (about 50%) of breast cancer patients who are diagnosed in the premenopausal stage (less than 50 years of age). The only currently available predictive test for HR-positive Her2-negative EBC that has been validated in Indian patients is CanAssist Breast. If this test gives a score indicative of low risk (< 15.5), adjuvant chemotherapy will not increase the chance of metastasis-free survival and should not be given. This is applicable even during the ongoing COVID-19 pandemic.

Nicotine-induced proliferation of isolated rat pancreatic acinar cells: effect on cell signalling and function.

OBJECTIVES: The aim of the current study was to investigate whether nicotine treatment would induce the proliferation of isolated rat primary pancreatic acinar cells in culture by activating mitogen-activated protein kinase (MAPK) signalling and exocrine secretion. MATERIALS AND METHODS: A nicotine dose- and time-response curve was initially developed to determine the optimal dose and time used for all subsequent studies. Proliferation studies were conducted by cell counting and confirmed further by bromodeoxyuridine (BrdU) incorporation and flow cytometry assays. MAPK signalling studies were conducted by Western blot analysis. Localization of ERK1/2 signals, with or without nicotine and the MAPK inhibitor, was visualized by immunofluorescence. RESULTS: Nicotine treatment caused dose-dependent activation of extracellular signal-regulated kinases (ERK1/2), the maxima occurring at 100 micro m and at 3 min after treatment; the response was suppressed by the ERK1/2 inhibitor. Maximal nicotine-induced cell proliferation occurred at 24 h, and UO126-treatment significantly reduced this response. Exposure of cells to 100 microm nicotine for 6 min significantly enhanced both baseline and cholecystokinin-stimulated cell function, and these effects were not affected by treatment with the inhibitor of ERK1/2 but were suppressed by mecamylamine, a nicotinic receptor antagonist. CONCLUSIONS: Our results suggest that nicotine treatment induced cell proliferation of isolated pancreatic acinar cells and that this is coupled with the activation of MAPK signalling with no effect on its function. Hence, in primary cells, the mechanism of induction and regulation of these two processes, cell proliferation and cell function, by nicotine treatment are independent of each other.

Minimally invasive radioguided surgery for parathyroid adenomas (MIRP).

BACKGROUND: Parathyroid adenoma is the most common cause of primary hyperparathyroidism. Conventional surgical management includes bilateral neck exploration with removal of the adenoma(s) and biopsy of one of the other glands with visualization of all glands. It is associated with a risk of permanent hypoparathyroidism. Radioguided excision of parathyroid adenoma is a widely accepted technique which provides accurate localization and complete excision of the lesion with low morbidity. We report our experience with this technique. METHODS: We performed radioguided excision of parathyroid adenomas in 15 patients. All of them had preoperative localization of the adenoma using a dual tracer, dual phase 99mTc-Sestamibi scan. A dose of 8-10 mCi of 99mTc-Sestamibi was injected intravenously 2 hours before surgery. Under local anaesthesia, surgical excision of the lesion was done after localizing it using a hand-held gamma probe. Complete excision was confirmed by frozen. section of the excised lesion and an intraoperative quick parathormone assay. RESULTS: The 99mTc-Sestamibi scan revealed an increased uptake by the adenoma in all patients and complete excision was possible in all the patients. Frozen section confirmed the diagnosis and the quick parathormone assay (within 15 minutes) revealed a drop in parathormone levels to < 50% after excision in all of them. Three patients developed hypocalcaemia postoperatively and were treated with intravenous calcium supplementation. At a follow up of 2-29 months, all the patients were normocalcaemic. The renal functions improved in 2 of 6 patients who had renal failure. CONCLUSION: Minimally invasive radioguided excision of parathyroid adenomas is a simple, safe and effective technique associated with a low morbidity and can be done as a day-care procedure.

Comparison study of quality of life in advanced lung cancer patients on tyrosine kinase inhibitor and platinum doublet chemotherapy.

INTRODUCTION: Lung cancer is most common cause of cancer death in the world. Most of the patient are diagnosed in the late stages and receive only palliative treatment. The main objective of the palliative chemotherapy is to improve survival as well as the quality of life (QOL). QOL is the most neglected dimension of cancer care in developing countries like India. Palliative chemotherapeutic agent which has minimum toxicity and prolongs the survival of metastatic cancer patients is the need of the day. MATERIALS AND METHODS: In this study, 43 metastatic adenocarcinoma of lung patients of South Indian origin were enrolled. Twenty patients out of this 43 were epidermal growth factor receptor (EGFR) mutation positive and were started on tyrosine kinase inhibitor (TKI). Rest 23 patients were EGFR mutation negative and were started on various platinum-based doublet chemotherapy. QOL was measured using Cancer Institute QOL Questionnaire version 2 at the beginning of therapy and at the end of 3 months. RESULTS: Our study showed that metastatic lung cancer patients had average QOL at presentation. The QOL in patients on TKI improved compared to those on platinum doublet chemotherapy during the second assessment, but this improvement was statistically not significant. CONCLUSION: In this study, the metastatic lung cancer patients had an average QOL during initial presentation. Patients on TKI had a trend toward better QOL after 3 months of treatment compared to platinum doublet chemotherapy.

Hepatic gateways.

The intestinal mucosal barrier contributes to homeostasis by limiting systemic dissemination of microbes and toxins while allowing nutrients to pass through to the systemic circulation. In a recent issue of Science, Spadoni et al. demonstrated a novel mechanism to enable this selectivity: the existence of a gut-vascular barrier (GVB) as indicated by a series of studies on the interaction between murine and human intestine with Salmonella typhimurium species . They showed that (i) enteroglial cells and pericytes in contact with endothelial cells (ECs) form the GVB (ii) Salmonella typhimurium can penetrate it by a mechanism dependent on the pathogenicity island (Spi) 2-encoded type III secretion system and on decreased ß-catenin dependent signaling in gut endothelial cells. Understanding the GVB may provide new insights into the regulation of the gut-liver axis.

Effect of current density during electrodeposition on microstructure and hardness of textured Cu coating in the application of antimicrobial Al touch surface.

Copper is a well proven antimicrobial material which can be used in the form of a coating on the touch surfaces. Those coating can offer a good service as touch surface for very long time if only they possess good mechanical properties like scratch resistance and microhardness. In the present work the above mentioned mechanical properties were determined on the electrodeposited copper thin film; deposited on double zincated aluminium. During deposition, current density was varied from 2Adm(-2) to 10Adm(-2), to produce crystallite size in the range of 33.5nm to 66nm. The crystallite size was calculated from the X-ray peak broadening (Scherrer׳s formula) which were later confirmed by TEM micrographs. The scratch hardness and microhardness of the coating were measured and correlated with the crystallite size in the copper coating. Both characteristic values were found to increase with the reduction in crystallite size. Reduced crystallite size (Hall-Petch effect) and preferred growth of copper films along (111) plane play a significant role on the increase in the hardness of the coating. Further, TEM analysis reveals the presence of nano-twins in the film deposited at higher current density, which contributed to a large extent to the sharp increase of coating hardness compared to the mechanism of Hall-Petch effect. The antimicrobial ability of the coated sample has been evaluated against Escherichia coli bacteria and which is compared with that of commercially available bulk copper using the colony count method. 94% of E. coli cells were died after six hours of exposure to the copper coated surface. The morphology of the copper treated cells was studied using SEM.

Cell kinetics of erythroid colony-forming cells (CFU-E) studied by hydroxyurea injections and sedimentation velocity profile.

Cell kinetic characteristics of murine CFU-E were assessed by measuring femoral marrow CFU-E survival rates after injection of hydroxyurea at from 2 to 48 h before marrow collection. The results are compatible with the concept that the CFU-E represent a homogeneous non-selfsustaining population comprised of cells belonging to a single differentiation stage. Their cell cycle length was estimated to be 10 h. Marrow cells were also subjected to separation by velocity sedimentation, and the effect of preceding hydroxyurea injections on the distribution profile of CFU-E was ascertained. Variations in cell size of CFU-E were interpreted to represent CFU-E in various phases of their cell cycle. CFU-E in either G1, S or G2 at the time of their removal from the marrow were capable of forming erythroid colonies in vitro, but it appears that the CFU-E lost this capacity after completing their cell cycle within the marrow of the intact mouse.

An evaluation of the role of microenvironmental factors in the limitation of myelopoiesis in murine long-term bone marrow culture.

On a day-to-day or week-to-week basis, generation of colony-stimulating factor (CSF) by the adherent layer was identical in long-term bone marrow cultures initiated from young or old mice. CSF concentration increased as myelopoiesis failed, no decline in the generation of CSF following increased stimulation was seen, and donor age did not affect the responsiveness of myeloid cells to CSF. Following spontaneous failure of myelopoiesis, adherent layers originally initiated from young or old mice were equally able to support hematopoiesis following recharge. Thus no age-related decline in adherent layer function could be detected.

Effect of donor and culture age on the function of neutrophils harvested from long-term bone marrow culture.

Neutrophils harvested from the supernatants of long-term bone marrow cultures that were initiated from young and old mice were tested for their functional activity. Superoxide generation was measured, as was the secretion of the enzymes lysozyme, myeloperoxidase, and glucuronidase, both basally and following stimulation by phorbol myristate acetate. The function of the neutrophils harvested from cultures that were initiated from young mice was identical to that of cells derived from young mouse peritoneal cavities at the time of death. A minimal decline in function in culture-derived cells was seen over the 15-18 weeks of study. For each of the above measurements examined, neutrophils obtained from cultures initiated from old mice gave values significantly lower than those in neutrophils recovered from cultures initiated from young mice. For some parameters (superoxide generation, myeloperoxidase), the rate of decline in function was more rapid for neutrophils from old rather than young mouse cultures. For other measurements the rates of decline were equal. The results indicated adequate neutrophil function in long-term bone marrow culture for extended periods of time. They also demonstrated that the age-related diminution in neutrophil function observed in vivo persisted in in vitro culture.

Studies on the kinetics of the erythroid colony-forming cell.

The appearance, rate of formation, and morphologic characteristics of the erythroid colony-forming cell (CFU-E) was studied in control, exhypoxic polycythemic, and anemic mice. Polycythemia resulted in a significant reduction in CFU-E number and a lag time of approximately 8 h before colonies were visible in culture. Thereafter, the rate of formation of colonies, as assessed by the slope of the increase in their number, was identical to the control. The slower appearance of CFU-E in polycythemia was corrected by the injection of erythropoietin (Ep) 8 h prior to sacrifice, but an absolute increase in colony number only occurred when Ep was injected 24 h previously. Polycythemia also slowed the rate of colony maturation, so that at later culture times the colonies more frequently contained fewer cells and consisted of basophilic normoblasts. Anemia did not shorten the time of first appearance of colonies, but their rate of maturation was significantly more rapid than the control. These findings are best explained by a redistribution of CFU-E in various phases of the cell cycle when erythropoiesis is manipulated. Unit-gravity sedimentation studies suggested a greater fraction of CFU-E in long G1 or G0 in polycythemia. In addition, the kinetics of CFU-E development were identical to polycythemia in mice in which S and post-S-phase CFU-E were removed by hydroxyurea injection. It is likely that Ep stimulates CFU-E in all phases of the cell cycle. After stimulation by Ep, however, CFU-E in long G1 must move through the cell cycle prior to commencing cell division. This transit time of 8 h, which is unaffected by the Ep concentration in the medium, results in a lag in appearance and maturation of colonies derived from these cells. These results demonstrate that cellular variables are important in CFU-E kinetics in vitro and provide insights into factors affecting the regulation of normal erythropoiesis.

Stimulation of granulopoiesis by androgens without concomitant increase in the serum level of colony stimulating factor.

The marked enhancing effect of daily injections of 19-nortestosterone decanoate (19-ND) upon granulopoietic recovery of mice made neutropenic by a single dose of BCNU was studeid in regard to the possible regulatory role of CSF. Serum levels of CSF in BCNU-treated mice with or without 19-ND injection were not found to differ significantly from those in normal mice. BCNU treatment with or without 19-ND did not alter the CSF increases in response to endotoxin injection. Increases in dividing marrow granulocytes in the 19-ND mice were preceded by increases in their marrow CFC. The acceleration of granulopoietic regeneration was thus not mediated by the serum colony stimulating factor. It is attributed to a stimulatory effect of 19-ND on proliferation of CFC and/or CFU.

The quantitation of the granulocytic/macrophage committed progenitor cell (CFUc) in man and the mouse.

A method has been developed to quantitate the morphologically unrecognizable granulocytic/macrophage committed progenitor cell (CFUc). In man this is done by relating CFUc colonies cultured in vitro to marrow normoblast number determined by ferrokinetic measurements. In the mouse total hematopoietic precursors are determined by a radioiron dilution technique and CFUc are quantitated by calculating the percentage of colonies contained in the known number of nucleated cells plated. Like other hematopoietic precursors, CFUc numbers in 14 normal men and 10 normal mice were not significantly different, the mean being 0.85 and 1.4 x 10(7) CFUc/kg body weight, respectively. The validity of these values was substantiated by demonstrating an excellent correlation in the mouse between CFUc/kg body weight and CFUc/femur. In subsequent studies CFUc were quantitated in patients with lung cancer prior to and following aggressive combination chemotherapy when hematopoietic suppression was at a maximum. Compared to a mean value of 39 CFU/10(5) cells plated from chemotherapy the value 10 days later was significantly higher, the mean being 125/10(5) cells. That this reflected a relative change in the proportion of CFUc was suggested by the finding that 10 days after chemotherapy the mean value for CFUc/kg was 1.94 x 10(7) which was not significantly greater than a mean of 1.53 x 10(7) before treatment. These studies illustrate the advantages of expressing CFUc/kg body weight and indicate the usefulness of the approach in the evaluation of CFUc growth in a wide range of physiologic and pathologic conditions. In addition to better comparisons of results obtained in groups of individuals, multiple studies in single persons in whom hematopoiesis is not in a steady state will also be possible.

Stimulation of erythroblast formation in suspension cultures of murine marrow by a factor in normal mouse serum, and its relationship to erythropoietin.

Addition of normal mouse serum to liquid suspension cultures of murine marrow cells resulted in dose-related increases in the number of heme-synthesizing erythroid cells. Removal of late normoblasts and reticulocytes by differential immune lysis prior to culture greatly enhanced the difference in heme synthesis between cultures with or without added mouse serum, permitting detection of as little as 1 microliter/l.5 ml of medium. Cell counts on these cultures indicated that the mouse serum factor actually stimulated formation of late normoblasts through proliferation and maturation of their precursors. The effect required either the presence of Ep in the medium, or the presence in the to be cultured cell suspensions of proerythroblasts which had been induced by or had contact with Ep prior to collection of the marrow.

Long-term bone marrow culture as a model for host toxicity: the effect of methotrexate on hematopoiesis and adherent layer function.

Long-term bone marrow culture was used to examine the hematopoietic toxicity of the chemotherapeutic agent methotrexate. A dose-related suppression in myelopoiesis was seen when cultures were treated with 10(-3), 10(-4), or 10(-5) M methotrexate followed by folinic acid rescue. Differential counts revealed an initial decrease of postmitotic myeloid cells followed by mitotic precursors and myeloid colony-forming units (CFU-C). Myelopoiesis had disappeared by 7-10 days when 10(-3) M methotrexate was studied, by day 21 with 10(-4) M methotrexate, and by day 28 with 10(-5) M methotrexate. Although 10(-3) M methotrexate caused a predictable suppression in myelopoiesis, the effect of 10(-5) M methotrexate was more variable. In some studies this dose caused significant suppression of myelopoiesis, whereas in others less suppression occurred. This provides some evidence for varying host susceptibility to drug. Microenvironmental (adherent layer) cells were also affected by methotrexate. An increase in proliferation of these cells occurred in proportion to the dose of methotrexate added to culture. Methotrexate did not affect the ability of the adherent cells to produce colony-stimulating activity (CSA), but high doses did prevent the layer's ability to support the proliferation of adherent cell-free marrow. These results indicate that long-term bone marrow culture can be used to successfully predict and define chemotherapeutic host toxicity.

Evaluation of the effect of age on hematopoiesis in the C57BL/6 mouse.

The effect of age on hematopoiesis was studied in young (six-month) and old (24-month) C57BL/6 mice. In addition, studies were performed on very old (42-month) mice, housed either singly or in groups of five animals per cage. Although a reduction in hematocrit was found in the older mice, red cell mass was normal in both old and very old single-caged mice. A detailed evaluation of erythropoiesis that included plasma- and erythron-iron turnover (PIT and EIT), red cell survival, and quantitation of the marrow erythroid progenitor and differentiated cells demonstrated no age-related change in single-caged animals. Similarly, quantitation of marrow myeloid precursors was identical in these groups. These results indicate that no age-related change in basal hematopoiesis can be demonstrated even in animals approaching maximal life expectancy. When very old mice were routinely housed in groups of five per cage, however, a decrease in hematocrit was found which was accompanied by significant alterations in hematopoiesis, including reductions in total differentiated erythroid cells, erythroid burst-forming units (BFU-E), erythroid colony-forming units (CFU-E), and colony-forming-unit culture (CFU-C) levels. It is likely that in very old animals, group housing constitutes a sufficient stress to compromise hematopoiesis. These findings indicate that basal hematopoiesis is unaltered by aging, although the bone marrow's reserve capacity is markedly compromised.

Evidence suggesting a negative regulatory role for macrophages in murine erythropoiesis in vivo.

Increasing the rate of erythropoiesis in C57BL/6 mice, either by hypoxia or by the injection of recombinant erythropoietin (Epo), resulted in significant reductions in marrow macrophage number, as assessed by flow cytometry employing the monoclonal antibody against the macrophage antigen Mac-1 and by histologic determination of reductions in the number of marrow esterase-positive cells. This decline was paralleled by decreases in marrow colony-forming unit-macrophage (CFU-M) and colony-forming unit-granulocyte/macrophage (CFU-GM) number. The intramedullary concentration of the cytokines interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor-alpha (TNF-alpha), which are produced by macrophages, was also reduced. Cessation of erythropoiesis was associated with increases in macrophage number, CFU-M and CFU-GM colony number, and IL-1 alpha concentrations. Increased erythropoiesis resulted in reductions in number of burst-forming unit-erythroid (BFU-E) colonies, which were less sensitive to suppression by macrophages as evidence by less increase in colony number when macrophages were removed from the marrow before in vitro BFU-E culture. BFU-E colony number was suppressed less when IL-1 alpha and TNF-alpha were added to cultures obtained from animals with stimulated erythropoiesis. Compared to controls, BFU-E number and suppression by macrophages increased significantly when erythropoiesis was reduced. These observations provide compelling evidence for a regulatory role for macrophages in normal erythropoiesis in vivo, presumably acting as a negative balance to the stimulatory effects of Epo.

A micromethod for long-term in vitro culture of bone marrow cells.

A micromethod to study long-term culture of bone marrow cells in vitro has been developed. Using Linbro wells one-fourteenth of the cells and medium usually required for culture in a flask are plated. Thus 40-50 cultures from a single mouse can be studied at any one time. Adherent-layer formation and supernatant cell recovery were very similar when Linbro -well cultures were compared with standard large-flask cultures. In the microculture system, supernatant and adherent-layer cell numbers increased following medium change reaching a maximum 3-4 days later and decreasing by day 7. Supernatant cells were in equilibrium with those of the adherent layer as cell numbers in both compartments prior to and following medium change fluctuated identically. Following medium change, a significant increase in myeloblasts occurred at 24 h and promyelocytes and myelocytes increased 48-72 h later. In contrast no discernible pattern in daily CFU-C production was detected. These findings provide insight into the cellular kinetics of long-term marrow culture and highlight the usefulness of this method to study hematopoiesis at frequent intervals.