Tissue Culture Flasks Research Articles

Promoting and accelerating muscle regeneration through cell therapy in a mouse model

ObjectivesSkeletal muscle injuries and disorders are universal clinical challenges with direct and indirect mechanisms and notable residual effects, such as prolonged, intense pain and physical disability. Stem cells, an innovative tool for cell therapy for musculoskeletal disorders, specifically promote skeletal muscle regeneration. This study was aimed at investigating the use of mesenchymal stem cells (MSCs) and their differentiated myocytes as a cell-based therapy to promote regeneration in damaged or diseased skeletal muscle. MethodsBone marrow mesenchymal stem cells (BM-MSCs) were isolated from the bone marrow of adult mice and grown in tissue culture flasks. The BM-MSCs were positive for CD90 and CD105, and negative for CD45 and CD34. These cells were induced with specific differentiation medium in vitro to differentiate into a skeletal muscle cell lineage over 7 days. Skeletal muscle differentiation was characterized according to morphology through hematoxylin and eosin staining, and scanning electron microscopy. Immunostaining for Myf-6, myosin heavy chain (MHC), and desmin—specific factors for skeletal muscle development—was performed to confirm skeletal muscle differentiation. An in vivo study in a muscle injury model was used to evaluate cell therapy based on naïve stem cells and differentiated myocytes. ResultsCultured mouse BM-MSCS were positive for CD90 and CD105, and negative for CD45 and CD34. These cells developed into skeletal muscle with strong skeletal muscle differentiation potential, as confirmed by immunohistochemistry for the markers Myf6, MHC, and desmin. The differentiated myocytes showed better repair enhancement than undifferentiated stem cells after transplantations into a mouse model of skeletal muscle atrophy. ConclusionsMyocytes derived from BM-MSCs may be incorporated into muscular atrophy treatment as a biological strategy for managing skeletal muscle diseases and injuries, thus advancing cell-based clinical treatments.

Bringing cell biology into classroom: tips to culture and observe skeletal muscle cells in high school and college

Watching living cells through a microscope is much more exciting than seeing pictures of cells in high school and college textbooks. However, bringing cell cultures into the classroom is challenging for biology teachers since culturing cells requires sophisticated and expensive instruments such as a CO2 incubator and an inverted phase-contrast microscope. Here, we describe easy and affordable methods to culture and observe skeletal muscle cells using the L-15 culture medium, tissue culture flask, standard dry incubator, standard upright microscope, and modified Smartphone microscope. Watching natural living cells in a “Do-It-Yourself (DIY)” way may inspire more students’ interest in cell biology.

Effects of p300 inhibition on prostate cancer cell growth

Background: Radiation is a central component of many anti-cancer therapy regimens, utilized for more than half of all cancer patients. Despite advances in the delivery and success of radiation treatment, many individuals express, or will develop, radioresistance, a major contributor to treatment failure and malignant progression. While the etiology of radioresistance is complex, it may be mediated in part by expression of p300, a histone acetyltransferase known to facilitate DNA repair. We hypothesized that inhibition of p300 would increase radiosensitivity of prostate cancer cells in vitro. Methods: Human prostate adenocarcinoma (PC-3) cells were grown in tissue culture flasks incubated at 37° C with 5% CO2. Cells were randomized to receive 1 μM CCS 1477, a selective inhibitor of p300, or vehicle for 1 hour prior to a single treatment of 0 Gy (NR+ or NR-, respectively) or 2 Gy radiation (R+ or R-, respectively). Four hours after radiation, cells were seeded into 100 mm diameter tissue culture dishes and incubated for 10 days. Cells were then stained, and colonies > 50 cells were counted to determine clonogenic survival. Cell survival was corrected for plating effciency and group comparisons were made using a two-way ANOVA. Results: Results were determined from the average of 3 separate experiments. Compared to non-radiated controls (NR-), survival of R- was reduced to 47%, while survival of R+ was reduced to 32%. However, there was no significant difference between % survival of R- and R+. Conclusions: In vitro, 1 μM exposure to CCS 1477 does not reduce clonogenic survival in PC-3 cells subjected to 2 Gy radiation. The duration of exposure and concentration of CCS 1477 may not be adequate to elicit significant improvements in radiosensitivity of PC-3 cells to 2 Gy radiation. Future experiments will explore the effects of varying durations of exposure and concentrations of CCS 1477. Additionally, it remains to be determined whether CCS 1477 improves radiosensitivity in other cancer cell lines (e.g., breast cancer). Johnson Cancer Research Center. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

In vitro Wound Healing Effect of a Siddha Formulation: Gandhaga Thailam

In vitro Wound Healing Effect of a Siddha Formulation: Gandhaga Thailam

Carbohydrates Are Associated with the Flowering Ability of Oncidesa Gower Ramsey ‘Honey Angel’

Oncidesa Gower Ramsey ‘Honey Angel’ is a cut flower crop of high economic value worldwide. The regulation of flowering is important for cut flower production scheduling. However, its flowering transition mechanism is still unclear. Oncidesa usually flowers at the end of the growth cycle for each pseudobulb; this timing is probably related to carbohydrate accumulation. During this study, we investigated the carbohydrates in the pseudobulbs from juvenile plants to adult plants and compared the carbohydrates in flowering and nonflowering adult plants. The current pseudobulb and back pseudobulbs of the plants at 0, 0.5, 1.0, 1.5, and 2.0 years after having been moved out of the tissue culture flask were collected. The first pseudobulb formed at 0.5 years, and plants had fulfilled four growth cycles and flowered at 2.0 years. Each successive current shoot grew larger and the back shoot number progressively increased after each new growth cycle. The concentration of total soluble sugars in the current shoot increased from 5.5% of dry weight at 0.5 years to 20.2% of dry weight at 1.5 years. Conversely, the starch concentration decreased in the current pseudobulb as the plants matured. The starch concentration in the back pseudobulbs did not change when the plant grew a new shoot. The starch concentrations in the back pseudobulbs ranged from 33.2% to 57.5% of dry weight, but the combined content of starch in all of the back pseudobulbs increased significantly from 168 mg at 0.5 years to 4608 mg at 2.0 years because of the increasing number of back shoots. The starch in the first back pseudobulb of the nonflowering adult plants accounted for 18.0% of dry weight, which was lower than that of the flowering plants (48.3%). There was no significant difference in total soluble sugars in the current pseudobulb of the nonflowering and flowering plants. Overall, we revealed that the increase in the back shoot number increased the total amount of reserve carbohydrates as the plant reached reproductive maturity. A low starch level was observed in nonflowering adult plants. In both cases, flowering plants had higher starch storage in the back pseudobulbs, suggesting that carbohydrates might regulate the flowering of Oncidesa Gower Ramsey ‘Honey Angel’.

Virus Isolation of Epizootic Hemorrhagic Disease Virus in Cell Culture.

Virus isolation is used to assist in the diagnosis and confirmation of viral infections. Successful isolation of a virus is highly dependent upon the quality of starting material. Here we describe the preparation and isolation of epizootic hemorrhagic disease virus (EHDV) from blood and tissue samples in tissue culture flasks (TCFs) through the inoculation of susceptible cell lines including Vero, BHK, and KC cells.

Isolation and Culture of Human Umbilical Vein Endothelial Cells (HUVECs).

This protocol describes a simple and an economical method for isolation of endothelial cells from human umbilical vein. Umbilical cord is easily available postpartum following informed consent, and the method for its collection is noninvasive with few ethical concerns. Thus, umbilical vein is an ideal source for isolation of endothelial cells of human origin. Endothelial cells are isolated from umbilical vein by collagenase digestion. This is followed by their culture on extracellular matrix (ECM) protein coated tissue culture flasks in presence of endothelial cell growth medium. In the last few decades, human umbilical vein endothelial cells (HUVECs) have come to be regarded as a standard model system by vascular biologists to understand general principles of endothelial cell biology and dysfunction. Here, we describe isolation, culture, as well as validation of HUVECs.

Comparative Analysis of MEM, RPMI 1640 Media on Rabies Virus Propagation in Vero Cells and Virus Quantification by FAT

Rabies disease can be preventable through vaccination is the only way for effective control and the vaccine is administered as post exposure prophylaxis method (PEP) along with rabies immunoglobulin. For the preparation of the vaccine, the high titre rabies virus has to be propagated in suitable host system like Vero cells. In this study, the virus was propagated in tissue culture flask in two different tissue culture media namely MEM and RPMI-1640, keeping Vero cell line as the host system. This preliminary study is to find the media that yields a better viral titre as high, the influence of harvesting intervals on viral titre for the reason that the vaccine yield is directly proportional to the viral titre during the virus propagation stage and leads to cost effective vaccine. The Vero cell was revived from passage 154 and the part of cells were subjected for adaptation in RPMI-1640 media with gradual media replacement. The passaging of RPMI 1640 media cells were continued until it reaches the equal cell count of MEM (p-158). The confluent monolayer of Vero cells was maintained in the same passage level with appropriate media. The cell count of MEM media, RPMI-1640 were 10.28 x 106 and 10.35 x 106 respectively in 25cm2 tissue culture flasks. The Vero cells were sub-cultured in 175 cm2 tissue culture flasks infected with 0.2 MOI (Multiplicity of Infection) of virus and the viral titration was estimated through FAT test (Fluorescent Antibody Test). The highest viral titre obtained from RPMI-1640 media batch (10-6.125/ml) and MEM media batch (10-6.25 /ml). The two days interval viral harvests shows (Batch 1 & 3) the viral titre log ranged between 10-4.375 to 10-5.875 and three days interval harvests (Batch 2 & 4) the viral titre log ranged between 10-3.750 to 10-6.250 per mL.

Antibodies from serum and CSF of multiple sclerosis patients bind to oligodendroglial and neuronal cell-lines.

Multiple sclerosis is a highly complex and heterogeneous disease. At the onset it often presents as a clinically isolated syndrome. Thereafter relapses are followed by periods of remissions, but eventually, most patients develop secondary progressive multiple sclerosis. It is widely accepted that autoantibodies are important to the pathogenesis of multiple sclerosis, but hitherto it has been difficult to identify the target of such autoantibodies. As an alternative strategy, cell-based methods of detecting autoantibodies have been developed. The objective of this study was to explore differences in the binding of antibodies from sera and CSF of multiple sclerosis patients and controls to oligodendroglial and neuronal cell-lines, related to antibody type, immunoglobulin (IgG/IgM), matrix (serum/CSF) and disease course. The oligodendroglial and neuronal cell-lines were expanded in tissue culture flasks and transferred to 96-well plates at a concentration of 50 000 cells/well followed by fixation and blocking with bovine serum albumin. Sera and CSF samples, from healthy controls and multiple sclerosis patients, were incubated with the fixed cells. Epitope binding of immunoglobulins (IgG and IgM) in sera and CSF was detected using biotinylated anti-human IgM and IgG followed by avidin conjugated to horseradish peroxidase. Horseradish peroxidase activity was detected with 3,3',5,5'-tetramethylbenzidine substrate. Serum from 76 patients and 30 controls as well as CSF from 62 patients and 32 controls were investigated in the study. The binding was similar between clinically isolated syndrome patients and controls, whereas the largest differences were observed between secondary progressive multiple sclerosis patients and controls. Antibodies from multiple sclerosis patients (all disease course combined) bound more to all investigated cell-lines, irrespectively of matrix type, but binding of immunoglobulin G from CSF to human oligodendroglioma cell-line discriminated best between multiple sclerosis patients and controls with a sensitivity of 93% and a specificity of 96%. The cell-based enzyme linked immunosorbent assay (ELISA) was able to discriminate between multiple sclerosis patients and controls with a high degree of accuracy. The disease course was the major determinant for the antibody binding.

Inhibition Effect of Cell-Free Supernatants of Environmental Acanthamoeba Strains on the Viability of Metastatic Cell-Lines

It is known that some of the therapeutic agents against cancer cells are isolated from natural sources such as plants and animals. However, due to increasing drug resistance, studies on the discovery of new sources are needed. In this study, it was aimed to investigate the inhibition effects of four native Acanthamoeba strains on different cancer cell lines (MDA-MB-231, PC3, MAT-LyLu). 3T3 cells were used as normal cell line. All strains were recultured by using non-nutrient agar spread by heat-inactivated Escherichia coli ATCC 25922. A.castellanii ATCC 50373 was used as the standard strain. Molecular identification of the native Acanthamoeba isolates was done by polymerase chain reaction (PCR) and DNA sequence analysis using specific primer pairs (P-FLA-F, P-FLA-R, JDP-F, JDP-R). Axenic cultures of all strains were obtained in 25 cm2 tissue culture flasks and in peptone yeast extract glucose (PYG) medium. In order to investigate the effect of cell-free supernatants obtained from axenic cultures on cancer cell lines and 3T3 cell viability, MTT method was applied using different concentrations of cell-free supernatants (1%, 2%, 5%, 10%, 15%). It was determined that the viability of 3T3 cells was not affected by any Acanthamoeba cell-free supernatants (p≤ 0.05). All of the samples tested were found to have a significant inhibitory effect (p<0.05) on the viability of PC3 and MAT-LyLu cells (human and rat prostate cancer cell line). However, none of the samples had an inhibitory effect on the viability of MDA-MB-231 (breast cancer cell-line). Two native Acanthamoeba cell-free supernatants showed higher inhibitory potency (28% and 21.9%) at 2% concentration against PC3 cells compared to the reference strain (16%). Similarly, the same Acanthamoeba samples were also shown to have a better inhibition potential on the viability of MAT-LyLu cells than the reference strain. It was found that the inhibitory potential of Acanthamoeba cell-free supernatants may not be related to proteins and proteases. The results obtained from this study showed that Acanthamoeba species living in the aquatic environment isolated from our country have a potential inhibitory effect against the tested cancer cell lines. In addition to plants and animals, Acanthamoeba cell-free supernatants can also be a source for natural therapeutic substances that act against cancer cells. However, it is necessary to carry out new studies using more strains in order to detect strains with higher inhibitory effects.

An optimized HEK293T cell expansion protocol using a hollow-fiber bioreactor system.

Viral vectors are commonly used to introduce genetic material into cells to modify cell function for a variety of purposes. Manufacture of those modified viruses may use a variety of cell types to generate high titers of viral particles; one of the most common being HEK293 cells. These cells have been modified into different lines aimed at satisfying specific use cases. HEK293T cells, for example, have been modified to include the SV40 large T antigen. Efficient viral particle production by HEK293T cells requires the maintenance of favorable cell culture conditions during expansion and transfection. This protocol describes the use of the Quantum® hollow-fiber bioreactor (HFB) system for the automated expansion of HEK293T cells, and the results derived using the protocol described herein were not compared with those from tissue culture flasks or other expansion platforms, as the parameters described are unique to Quantum's hollow fiber cell expansion environment. The purpose of this protocol is to help users of Quantum to focus on relevant parameters of expansion in the HFB milieu and to provide guidelines for a successful expansion of HEK293T cells in the Quantum system. The steps provided have been optimized to reliably control environmental factors related to glucose, lactate, and pH. Data reflecting this consistency are provided along with procedural time points reflected in text and figure formats.

Testing 3D printed biological platform for advancing simulated microgravity and space mechanobiology research

The advancement of microgravity simulators is helping many researchers better understanding the impact of the mechanically unloaded space environment on cellular function and disfunction. However, performing microgravity experiments on Earth, using simulators such as the Random Positioning Machine, introduces some unique practical challenges, including air bubble formation and leakage of growth medium from tissue culture flask and plates, all of which limit research progress. Here, we developed an easy-to-use hybrid biological platform designed with the precision of 3D printing technologies combined with PDMS microfluidic fabrication processes to facilitate reliable and reproducible microgravity cellular experiments. The system has been characterized for applications in the contest of brain cancer research by exposing glioblastoma and endothelial cells to 24 h of simulated microgravity condition to investigate the triggered mechanosensing pathways involved in cellular adaptation to the new environment. The platform demonstrated compatibility with different biological assays, i.e., proliferation, viability, morphology, protein expression and imaging of molecular structures, showing advantages over the conventional usage of culture flask. Our results indicated that both cell types are susceptible when the gravitational vector is disrupted, confirming the impact that microgravity has on both cancer and healthy cells functionality. In particular, we observed deactivation of Yap-1 molecule in glioblastoma cells and the remodeling of VE-Cadherin junctional protein in endothelial cells. The study provides support for the application of the proposed biological platform for advancing space mechanobiology research, also highlighting perspectives and strategies for developing next generation of brain cancer molecular therapies, including targeted drug delivery strategies.

In vivo biosynthesis of long-chain polyunsaturated fatty acids by the euryhaline rotifer (Brachionus plicatilis)

In order to increase omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA) availability and to improve the design of rotifer's enrichment protocols for marine larvae production, the endogenous fatty acid (FA) metabolism of the marine rotifer, Brachionus plicatilis was determined. To this purpose, the in vivo ability of this species to incorporate, esterify into the different lipid classes and to elongate/desaturate unsaturated FAs was investigated. Rotifers were incubated in 75 cm2 tissue culture flasks, at a density of 75,000 rotifers per incubation, for 4 h. Incubations (n = 4) were performed at 21 °C, with 0.3 μCi of [1-14C]FAs including C18 FAs (18:1n-9, OA; 18:2n-6, LA; 18:3n-3, ALA) and LC-PUFA (20:4n-6, ARA; 20:5n-3, EPA; 22:6n-3, DHA). The present study demonstrates that rotifers possess an active metabolism over dietary FAs, being this species able to produce LC-PUFA from its C18 FA precursors. Interestingly, the action of a Δ12 desaturase that converts OA to LA and of a Δ15 desaturase over LA to produce ALA, seems to be also present, in agreement with previously reported functional characterization of ωx desaturases genes in this species. However, a globally low transformation rate was obtained, in addition to a lower incorporation of DHA into rotifer tissues compared to the other incubated substrates. The aforementioned factors, together with a competitive esterification pattern of DHA, LA and ALA into phosphatidylcholine (PC) or of EPA and ARA into phosphatidylinositol (PI), may give rise to rotifers with inadequate essential FA ratios for marine larvae nutrition, if ignored or incorrectly handled in the lipid enrichment process of this live prey species.

Evaluation of Fatty Acid Oxidation in Cultured Bone Cells.

Bone formation by differentiating osteoblasts is expected to require significant energetic input as these specialized cells must synthesize large extracellular matrix proteins that compose bone tissue and then concentrate the ions necessary for its mineralization. Data on the metabolic requirements of bone formation are emerging rapidly. While much remains to be learned, it is expected that derangements in the intermediary metabolism contribute to skeletal disease. Here, a protocol is outlined to assess the capacity of osteoblastic cells to oxidize 14C-labeled fatty acids to 14CO2 and acid-soluble metabolites. Fatty acids represent a rich-energy reserve that can be taken up from the circulation after feeding or after their liberation from adipose tissue stores. The assay, performed in T-25 tissue culture flasks, is helpful for the study of gene gain or loss-of-function on fatty acid utilization and the effect of anabolic signals in the form of growth factors or morphogens necessary for the maintenance of bone mass. Details on the ability to adapt the protocol to assess the oxidation of glucose or amino acids like glutamine are also provided.

So, you want to create a frog cell line? A guide to establishing frog skin cell lines from tissue explants

Skin is an important interface with the external environment and investigating amphibian skin cell biology will improve our understanding of how environmental factors such as pathogens and pollutants are contributing to global amphibian declines. There is a critical need for in vitro systems to facilitate conservation research in model and non-model amphibians and the creation of new amphibian cell lines will play a significant role in reducing or even replacing the use of live animals for in vivo studies by providing an in vitro alternative. Here, we detail an adapted protocol for the generation of spontaneously arising cell lines from frog skin tissues, without the need for immortalization steps. Expanding the amphibian invitrome will foster and expedite new research in amphibian gene function, cellular responses, host-pathogen interactions, and toxicology. The following customizations to traditional tissue explant generation procedures have facilitated the successful generation of adherent skin epithelial-like cell lines from Xenopus laevis and can be further adapted for use with different frog species, such as Rana sylvatica, and different tissues:•Osmotic adjustment of culture medium and solutions for different amphibian species.•Use of small tissue explants, instead of enzymatic digestion of tissues, and gentle spotting of these tissue explants onto the growth surface of tissue culture flasks to promote better tissue adherence.•Partial replacement of medium to allow accumulation of potential endogenous growth factors in cultures.

Radiation Resistance of C57Bl/6 Mouse Bone Marrow Stromal Cell Lines Induced By Apoptosis Inhibitor JP4-039 or Necroptosis Inhibitor Necrostatin-1, but Not By Simultaneous Administration of Both Mitigators

Introduction: Necroptosis is a TNFa activated, caspase-independent cell death pathway that involves active disintegration of mitochondrial, lysosomal and plasma membranes (Vandenabeele, et al, Nat Rev Mol Cell Biol., 2010 Oct; 11(10):700-14). We measured clonogenic radiation survival of C57/Bl6 mouse bone marrow stromal cell lines in the presence of a mitochondrial targeted inhibitor of apoptosis (JP4-039), an inhibitor of necroptosis (Necrostatin-1) or both drugs.Materials and Methods: C57BL/6 mouse long term bone marrow cultures (LTBMCs) were established from adult female C57Bl/6 mice. Bone marrow stromal cell lines were derived from the adherent layers of LTBMCs. Radiosensitivity of C57Bl/6 bone marrow stromal cell lines and fresh bone marrow CFU-GEMM (semisolid medium with added hemopoietic growth factors) was measured in clonogenic radiation survival curves. Stromal Cells were irradiated to doses of 0 to 8 Gy, plated in T-75 tissue culture flasks, incubated for 8 days at 37oC, stained with crystal violet and colonies of greater than 50 cells counted at day 7. CFU-GEMMs from fresh bone marrow were scored at day 14. Both cultured cell populations were tested with addition of radiation mitigators: JP4-039 (10µM), Necrostatin-1 (40µM), or both drugs added to culture medium 30 minutes post irradiation. C57BL/6 mouse LTBMCs generated hemopoietic cells for 20 weeks. Marrow stromal cell lines were derived from 20 week old hemopoieticly-inactive culture adherent layer cells. Statistical analysis was performed using Student's t-test, with a two-tail p-value < 0.05 considered significant.Results: Radiation survival curves showed that C57Bl/6 fresh bone marrow CFU-GEMM and stromal cell lines were comparable to those for other mouse strains (CFU-GEMM Do = 1.67 ± 0.02, stromal cells Do=1.43 ± 0.14). Both fresh marrow CFU-GEMM and stromal cells (ň = 5.26 ± 0.72) were radioresistant when grown in presence of JP4-039 (ň = 8.85 ± 1.23, p = 0.012) or necrostatin-1 (n=7.97 ± 1.12, p = 0.024). Addition of both JP4-039 and Necrostatin-1 (ň = 5.79 ± 1.44) at the same time after irradiation (30 min) did not improve clonogenic survival of either fresh marrow CFU-GEMM or stromal cell lines.Conclusions: Both freshly explanted CFU-GEMM marrow hematopoietic progenitor cells and LTBMC derived marrow stromal cell lines display radioresistance when treated with each of two radiation mitigators that target different cell death pathways. While each drug delivered independently induced radioresistance, combined delivery of JP4-039 and Necrostatin-1 at 30 min after irradiation did not. Thus, the timing of delivery of drugs that target different cell death pathways is likely critical in designing effective protocols for radiation mitigation. DisclosuresNo relevant conflicts of interest to declare.

Utilization of Discarded Surgical Tissue from Ultrasonic Aspirators to Establish Patient-Derived Metastatic Brain Tumor Cells: A Guide from the Operating Room to the Research Laboratory.

Patient-derived cells from surgical resections are of paramount importance to brain tumor research. It is well known that there is cellular and microenvironmental heterogeneity within a single tumor mass. Thus, current established protocols for propagating tumor cells in vitro are limiting because resections obtained from conventional singular samples limit the diversity in cell populations and do not accurately model the heterogeneous tumor. Utilization of discarded tissue obtained from cavitron ultrasonic surgical aspirator (CUSA) of the whole tumor mass allows for establishing novel cell lines in vitro from the entirety of the tumor, thereby creating an accurate representation of the heterogeneous population of cells originally present in the tumor. Furthermore, while others have described protocols for establishing patient tumor lines once tissue has arrived in the research lab, a primer from the operating room (OR) to the research lab has not been described before. This is integral, as basic research scientists need to understand the surgical environment of the OR, including the methods utilized to obtain a patient's tumor resection, in order to more accurately model cancer biology in laboratory. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Establishment of brain tumor cell lines from patient-derived CUSA samples: processing brain tumor sample from the OR to the lab Support Protocol 1: Sterilization of microsurgical tools in preparation for dissection Support Protocol 2: Collagen coating of tissue culture flasks Basic Protocol 2: Selection of tumor cells in vitro Support Protocol 3: FACS sorting tumor sample to isolate cancer cells from heterogeneous cell population.

Effects of Heat Therapy on Prostate Cancer Cells Subjected to 4 Gy Radiation

Background Despite many advancements in treatment options, radiation therapy remains a common treatment for individuals diagnosed with prostate cancer. However, radiation resistance presents a significant challenge for many patients. Due to its numerous effects on cancer cell growth and survival, heat therapy has been studied as a potential mechanism to improve radiation efficacy. However, the modality of heat therapy varies widely between studies, and the majority of heat therapy research relies on non-physiological temperatures (>43º C). We hypothesized that mild temperature hyperthermia would improve the efficacy of radiation of prostate cancer cells in vitro. Methods Clonogenic survival was assessed using human prostate cancer (PC-3) cells in vitro. Cells were grown in tissue culture flasks maintained in a 37º C incubator with 5% CO2 and randomized into 3 groups: normothermic (NT; n=8), acute hyperthermia (HTA; n=8), and chronic hyperthermia (HTC; n=8). NT cells were maintained in a normal incubator until radiation. For heat treatments, HTA cells were heated to 41.0º C in a separate incubator for 1 h immediately prior to radiation. HTC cells were heated to 41.0º C in a separate incubator for 1 h each session, for a total of 3 heat treatments, each separated by 48 h, with the final heat treatment occurring 24 h prior to radiation. Immediately following a single dose of 4 Gy radiation, each group of cells were counted and plated into 60 mm culture dishes at densities of both 500 cells/plate and 1000 cells/plate, with 5 replicates plated per density. Following 8 days of incubation, cells were fixed and stained and colonies of greater than 50 cells were counted. Results The survival fractions were determined from the average of the two plating densities. Clonogenic cell survival was significantly reduced in HTA vs. NT (11.6% ± 2% vs. 19.3% ± 1%, p<0.05) and in HTC vs. NT (8.3% ± 2% vs. 19.3% ± 1%, p<0.05). Cell survival between HTA and HTC was not different (p>0.05). Conclusions These results suggest that both acute and chronic mild hyperthermia improve the efficacy of 4 Gy radiation in PC-3 cells. While exact mechanisms are yet to be elucidated, it is thought that hyperthermia at lower temperature induces DNA damage, inhibits DNA damage repair, or alters cell cycle progression, which individually or in combination with one another, may potentiate the effects of radiation. Mild temperature hyperthermia prior to radiation may be an effective neo-adjuvant to increase the radiosensitivity of prostate cancer cells.

Effects of Apigenin, Luteolin, and Quercetin on the Natural Killer (NK-92) Cells Proliferation: A Potential Role as Immunomodulator

Cancer can be classified as a fourth leading cause of death in Malaysia. There is a continuous effort by scientists in finding alternative cure to cancer due to the known side effects of chemotherapy and radiation therapy as well as recurrences. One of the latest methods to kill cancerous cells is by using immune cells known as natural killer (NK) cells. Flavonoids such as flavone and flavonol are also known for their antioxidant, anti-inflammatory, immunomodulatory and anticancer properties. This study was carried out to determine the role of flavonoid compounds of apigenin, luteolin, and quercetin to facilitate the growth of NK-92 cells. NK-92 cell line was grown in tissue culture flasks containing α- Minimum Essential Medium (MEM) medium enriched with L-glutamine, 12.5% fetal bovine serum, 12.5% horse serum, 0.2 mM myo-inositol, 0.02 mM folic acid, and 100 - 200 U/mL recombinant interleukin 2 (IL-2). The cell viability was determined via trypan blue staining where the cells were manually counted by a haemocytometer. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the cell viability of NK-92 cells after treatment with apigenin, luteolin, and quercetin. Results showed a dose-dependent proliferative effects of apigenin, luteolin, and quercetin on the proliferation of NK-92 cells showing the highest percentage of proliferation at 100 μg/mL for all compounds (*P &lt; 0.05). However, exceeding the dose of 100 μg/mL had resulted in a decline of cell proliferations percentage. Based on these findings, flavonoid compounds comprising apigenin, luteolin and quercetin were able to induce proliferative effects on NK-92 cells.

Monte Carlo simulation and dosimetry measurements of an experimental approach for in vitro HDR brachytherapy irradiation

Irradiation of tumor cell lines is a useful way to investigate the effects of ionizing radiation on biological molecules. We designed an easy and reproducible approach for in vitro experimental high dose rate brachytherapy, which was simulated by a Monte Carlo code and dosimetrically characterized by experimental methods to evaluate the correspondence between planned doses and doses absorbed by the cells. This approach is an acrylic platform containing T25 tissue culture flasks and multiwell tissue culture plates. It allows nine parallel needles carrying an 192Ir source to irradiate the adherent cells. The whole system composed of the acrylic platform, tissue culture flasks and 192Ir source tracking was simulated by the Monte Carlo N-Particle transport code (MCNPX). Dosimetric measurements were taken by well ionization chamber and radiochromic films. There was a slight difference, averaging from 2% to 7%, between the MCNPX results and film dosimetry results regarding uniform radiation created by the source arrangement. The results showed different values for planned and measured doses in each cell culture plate, which was attributed to the non-equivalent water material used and to the lack of full scattering coming from the top of the platform. This last contribution was different for each tissue culture plate and an individual dose correction factor was calculated. The dose correction factor must be applied to match the planned dose and the actual doses absorbed by the cells. The designed approach is an efficient tool for in vitro brachytherapy experiments for most commercial cell culture plates.