RESUMO
Although several technologies have been developed to isolate cells of interest from a heterogenous sample, clogging and impaired cell viability limit such isolation. We have developed the Enrich TROVO system as a novel, nonfluidic technology to sort live cells. The TROVO system combines imaging-based cell selection and photo-crosslinking of (gelatin methacrylate) gelMA-hydrogel to capture cells. After capture, cells are released by enzymatic digestion of the hydrogel and then retrieved for downstream analysis or further cell culturing. The system can capture cells with a recovery rate of 48% while maintaining 90% viability. Moreover, TROVO can enrich rare cells 506-fold with 93% efficiency using single step isolation from a 1:104 cell mixture, and can also capture one target cell from 1 million cells, reaching an enrichment ratio of 9128. In addition, 100% purity and 49% recovery rate can be achieved by a following negative isolation process. Compared to existing technologies, the TROVO system is clog-resistant, highly biocompatible, and can process a wide range of sample sizes.
Assuntos
Gelatina , Hidrogéis , Separação Celular , Metacrilatos , Engenharia TecidualRESUMO
Enriching target cell clones from diverse cell populations is vital for many life science applications. We have developed a novel method to rapidly and efficiently purify specific clonal cell populations from a larger, heterogeneous group using the Enrich TroVo system (Enrich Biosystems Inc., CT, USA). This system takes advantage of microfabrication and optical technologies by utilizing small hydrogel wells to separate desired cell populations and an innovative patching technique to selectively eliminate undesired cells. This method allows the isolation and growth of desired cells with minimal impact on their viability and proliferation. We successfully isolated and expanded clonal cell populations of desired cells using two model cells. Compared with fluorescence-activated cell sorting, Enrich TroVo system offers a promising alternative for isolating of sensitive, adherent cells, that is, patient-derived cells.
Assuntos
Citometria de Fluxo , Humanos , Citometria de Fluxo/métodos , Separação Celular/métodosRESUMO
Lynch syndrome (LS) is the most common hereditary form of colon cancer, resulting from a germline mutation in a DNA mismatch repair (MMR) gene. Loss of MMR in cells establishes a mutator phenotype, which may underlie its link to cancer. Acquired downstream mutations that provide the cell a selective advantage would contribute to tumorigenesis. It is unclear, however, whether loss of MMR has other consequences that would directly result in a selective advantage. We found that knockout of the MMR gene MSH2 results in an immediate survival advantage in human stem cells grown under standard cell culture conditions. This advantage results, in part, from an MMR-dependent response to oxidative stress. We also found that loss of MMR gives rise to enhanced formation and growth of human colonic organoids. These results suggest that loss of MMR may affect cells in ways beyond just increasing mutation frequency that could influence tumorigenesis.
Assuntos
Neoplasias Colorretais Hereditárias sem Polipose , Reparo de Erro de Pareamento de DNA , Humanos , Neoplasias Colorretais Hereditárias sem Polipose/genética , Mutação em Linhagem Germinativa , Células-Tronco , CarcinogêneseRESUMO
BACKGROUND: The dependence of tumor cells, particularly those originating in the brain, on glucose is the target of the ketogenic diet, which creates a plasma nutrient profile similar to fasting: increased levels of ketone bodies and reduced plasma glucose concentrations. The use of ketogenic diets has been of particular interest for therapy in brain tumors, which reportedly lack the ability to oxidize ketone bodies and therefore would be starved during ketosis. Because studies assessing the tumors' ability to oxidize ketone bodies are lacking, we investigated in vivo the extent of ketone body oxidation in 2 rodent glioma models. METHODS: Ketone body oxidation was studied using (13)C MR spectroscopy in combination with infusion of a (13)C-labeled ketone body (beta-hydroxybutyrate) in RG2 and 9L glioma models. The level of ketone body oxidation was compared with nontumorous cortical brain tissue. RESULTS: The level of (13)C-beta-hydroxybutyrate oxidation in 2 rat glioma models was similar to that of contralateral brain. In addition, when glioma-bearing animals were fed a ketogenic diet, the ketone body monocarboxylate transporter was upregulated, facilitating uptake and oxidation of ketone bodies in the gliomas. CONCLUSIONS: These results demonstrate that rat gliomas can oxidize ketone bodies and indicate upregulation of ketone body transport when fed a ketogenic diet. Our findings contradict the hypothesis that brain tumors are metabolically inflexible and show the need for additional research on the use of ketogenic diets as therapy targeting brain tumor metabolism.