Continuous Cell Culture Research Articles

Establishment of a cell culture from the frog Leptodactylus fuscus as a model for (eco)toxicological assays

Leptodactylus fuscus is a frog species widely distributed in the Neotropical region, occurring in several biomes, which makes it a potential biomonitor of environmental conditions. To advance the establishment of this species in this field, we developed a fibroblast-like cell line derived from the digits of the forelimbs (LFUfd) and evaluated its response to possible environmental stressors. An adult male L. fuscus was collected, anesthetized, and euthanized with eugenol. Digits from the forelimbs were extracted, decontaminated, and dissociated. Cells were plated and cultivated in vented flasks with DMEM/F12+GlutaMAX medium and 20% FBS at 28 °C. On the 15th passage, we made the chromosome preparations, where we placed them on slides and stained them with Giemsa for karyotype analysis. The cellular response to multiple stressors (Temperature, Hydrogen Peroxide, and Aluminum) was evaluated through the MTT assay. The temperature test evaluated two groups (28 °C and 37 °C for 24 h). The effect of hydrogen peroxide (H2O2) was analyzed by treatment for 1 h at 28 °C in different concentrations (control, 2 μM, 20 μM, 50 μM, 100 μM and 200 μM). The effects of aluminum have been tested at three concentrations (0.5 mg/L, 1 mg/L, and 10 mg/L). The karyotype obtained showed 22 chromosomes, according to the description of the species, although a small proportion of aneuploid cells was observed (∼7%). At 37 °C there was a reduction in cell survival and no effects on cell viability at the H2O2 concentrations tested; however, there was a decrease in cell viability when exposed to aluminum in all the concentrations tested.. The growth behavior and karyotype observed indicate the establishment of a continuous cell culture with a stable genome that responds to environmental changes.

3D Scaffold-Based Culture System Enhances Preclinical Evaluation of Natural Killer Cell Therapy in A549 Lung Cancer Cells.

Cell-based immunotherapies have emerged as promising cancer treatment modalities, demonstrating remarkable clinical efficacy. As interest in applying immune cell-based therapies to solid tumors has gained momentum, experimental models that enable long-term monitoring and mimic clinical administration are increasingly necessary. This study explores the potential of scaffold-based cell culture technologies, specifically three-dimensional (3D) extracellular matrix (ECM)-like frameworks, as promising solutions. These frameworks facilitate unhindered immune cell growth and enable continuous cancer cell culture. The three-dimensional (3D) cell culture model was developed using tailored scaffolds for natural killer (NK) cell culture. Within this framework, A549 lung cancer cells were cocultured with NK cells, allowing real-time monitoring for up to 28 days. The expression of critical markers associated with anticancer drug resistance and epithelial-mesenchymal transition (EMT) was evaluated in cancer cells within this 3D culture context. Compared to conventional 2D monolayer cultures, this 3D scaffold-based culture revealed that solid tumor cells, specifically A549 cells, exhibited heightened resistance to anticancer drugs. Additionally, the 3D culture environment upregulated the expression of EMT markers namely vimentin, N-cadherin, and fibronectin, while NK and zEGFR-CAR-NK cells displayed anticancer effects. In the two-dimensional (2D) coculture, only zEGFR-CAR-NK cells exhibited such effects in the 3D coculture system, highlighting an intriguing inconsistency with the 2D culture model, further confirmed by in vivo experiments. This in vitro 3D cell culture model reliably predicts outcomes in NK immunotherapy experiments. Thus, it represents a valuable tool for investigating drug resistance mechanisms and assessing the efficacy of immune cell-based therapies. By bridging the gap between in vitro and in vivo investigations, this model effectively translates potential treatments into animal models and facilitates rigorous preclinical evaluations.

Biological active compounds from edible mushroom: Effect on hela cells

Background: Mushrooms are unique organisms rich in proteins, carbohydrates, vitamins, and minerals, which allows them to be used not only as a source of nutrition but also in cosmetics and medicine. The fungus P. ostreatus possesses two strong enzymatic complexes – lignin-decaying and cellulose-decaying. Objective: The research aimed to study the antitumor activity of treated mm-wave mycelial extracts of mushrooms on the HeLa cell line. Research on edible mushrooms against tumors is very actual and important for the field of functional food science and bioactive compounds field. Methods: The effect of the fungal culture extract on the cyto-photometric and morphometric indices of the HeLa cell line culture was studied according to the nature of proliferation and transcription of malignant tissues. Using high-resolution liquid chromatography, the amino acid composition of acid-soluble proteins in mycelial extracts of a mushroom culture treated with mm waves was studied. Results: Extracts of P. ostreatus mycelial culture significantly suppressed the growth of proliferative activity of human tumor cultures and completely suppressed the mitotic activity of continuous HeLa cell cultures to 72 hours. Our study revealed a significant increase in glutamic and aspartic acids in mycelial culture extracts treated by mm-waves. Conclusion: Based on HPLC analysis, we suggest polyfunctional peroxidase as the candidate for the role of biologically active compound in the composition of aqueous extracts of the mycelial culture. So, the polyfunctional peroxidase plays an active role in the therapeutic properties of the oyster mushroom. Keywords: anticancer activity, ant-inflammation activity, mm-waves, mushroom`s, mycelial extracts, HPLC of acid-soluble proteins, HeLa cell line.

The Effect of Quercetin on Oxidatıve Stress in IPNV-infected Cells of Rainbow Trout (Oncorhynchus mykiss)

In this study, the antioxidant effects and the free radical scavenging capacity of quercetin were investigated in IPNV-infected Rainbow Trout Gonad-2 (RTG-2) continuous cell culture. For this purpose, four different groups were formed (Control, IPNV, Quercetin, IPNV+Quercetin). Malondialdehyde (MDA), Total Antioxidant Status (TAS), Total Oxidant Status (TOS), Ceruloplasmin (Cp), and Glutathione Peroxidase (GPx) activities were measured at 0, 24, 48, 72, and 96 hours. According to the results of our study, there was a significant increase in TAS, TOS, and Cp results in the groups containing IPNV-infected cells. According to TAS and TOS measurements, there was a statistically significant increase both between groups and at different hours. When GPx results were analyzed, it was observed that there was no statistically significant change between the groups and between the measurement hours. When the MDA results were analyzed, it was observed that there was no change in the measurements made at different hours, while there were significant differences between the groups. It was observed that quercetin can minimize the damage caused by scavenging free radicals in the environment and thus provides an important development for fish farming and economy in cells infected with IPNV in the laboratory environment.

Multiple mechanisms contribute to acquired TRAIL resistance in multiple myeloma

Multiple Myeloma (MM) prognosis has recently improved thanks to the incorporation of new therapies to the clinic. Nonetheless, it is still a non-curable malignancy. Targeting cancer cells with agents inducing cell death has been an appealing alternative investigated over the years, as is the case of TRAIL, an agonist of DR4 and DR5 death receptors. This pathway, involved in apoptosis triggering, has demonstrated efficacy on MM cells. In this research, we have investigated the sensitivity of a panel of MM cells to this agent and generated TRAIL-resistant models by continuous culture of sensitive cells with this peptide. Using genomic and biochemical approaches, the mechanisms underlying resistance were investigated. In TRAIL-resistant cells, a strong reduction in cell-surface receptor levels was detected and impaired the apoptotic machinery to respond to the treatment, enabling cells to efficiently form the Death Inducing Signalling Complex. In addition, an upregulation of the inhibitory protein c-FLIP was detected. Even though the manipulation of these proteins was able to modify cellular responses to TRAIL, it was not complete, pointing to other mechanisms involved in TRAIL resistance.

Single-Use, Metabolite Absorbing, Resonant Transducer (SMART) Culture Vessels for Label-Free, Continuous Cell Culture Progression Monitoring.

Secreted metabolites are an important class of bio-process analytical technology (PAT) targets that can correlate to cell conditions. However, current strategies for measuring metabolites are limited to discrete measurements, resulting in limited understanding and ability for feedback control strategies. Herein, a continuous metabolite monitoring strategy is demonstrated using a single-use metabolite absorbing resonant transducer (SMART) to correlate with cell growth. Polyacrylate is shown to absorb secreted metabolites from living cells containing hydroxyl and alkenyl groups such as terpenoids, that act as a plasticizer. Upon softening, the polyacrylate irreversibly conformed into engineered voids above a resonant sensor, changing the local permittivity which is interrogated, contact-free, with a vector network analyzer. Compared to sensing using the intrinsic permittivity of cells, the SMART approach yields a 20-fold improvement in sensitivity. Tracking growth of many cell types such as Chinese hamster ovary, HEK293, K562, HeLa, and E. coli cells as well as perturbations in cell proliferation during drug screening assays are demonstrated. The sensor is benchmarked to show continuous measurement over six days, ability to track different growth conditions, selectivity to transducing active cell growth metabolites against other components found in the media, and feasibility to scale out for high throughput campaigns.

Opportunities and Perspectives for Three Dimensional Culture of Mesenchymal Stem Cell-Derived Exosomes.

Exosomes are nanosized extracellular vesicles (EVs) that participate in intercellular communication through surface proteins and the delivery of internal cargo. The exosomes have gained attention for their potential as disease biomarkers and therapeutic agents. The therapeutic ability of exosomes has been verified by copious previous studies. Effective methods for extensive clinical applications are being researched for exosome-based regenerative therapies, including the application of 3D cultures to enhance exosome production and secretion, which can resolve limited exosome secretion from the parent cells. Cell culture has emerged as a crucial approach for biomedical research because of its many benefits. Both well-established continuous cell lines and primary cell cultures continue to be invaluable for basic research and clinical application. Previous studies have shown that three-dimensional cultured exosomes (3D-Exo) improve therapeutic properties and yields compared with traditional culture systems. Since the majority of studies have focused on exosomes derived from mesenchymal stem cells (MSC-Exo), this review will also concentrate on MSC-Exo. In this review, we will summarize the advantages of 3D-Exo and introduce the 3D culture system and methods of exosome isolation, providing scientific strategies for the diagnosis, treatment, and prognosis of a wide variety of diseases.

Transcriptomic analyses of host-virus interactions during in vitro infection with wild-type and glycoprotein g-deficient (ΔgG) strains of ILTV in primary and continuous cell cultures.

Infectious laryngotracheitis (ILT) remains a significant concern for the poultry industry worldwide due to its impact on animal welfare and its substantial economic consequences. The disease is caused by the alphaherpesvirus, infectious laryngotracheitis virus (ILTV). This study investigated in vitro host-virus interactions of a glycoprotein G (gG) deletion mutant vaccine strain of ILTV (ΔgG ILTV), and its parent wild-type strain (CSW-1 ILTV). Inoculations were performed separately for the two strains of ILTV using both a primary (chicken embryonic kidney, CEK) and a continuous culture (leghorn male hepatoma, LMH) of chicken cells. Transcriptome analysis was performed at 12 hours post infection. Each cell-type displayed distinct effects on host and viral gene transcription, with a greater number of viral and host genes differentially transcribed in CEK cells and LMH cells, respectively. Both cell-types infected with either strain demonstrated enrichment of pathways related to signalling, and gene ontologies (GO) associated with chemotaxis. Infection with either strain upregulated both SOCS proteins and certain proto-oncogenes, which may contribute to prolonged viral persistence by promoting immunosuppression and preventing apoptosis, respectively. Patterns of gene transcription related to cytokines, chemokines, endosomal TLRs, and interferon responses, as well as pathways associated with histone acetylation, transport, and extracellular matrix organization were similar within each cell type, regardless of the viral strain. In CEK cells, GO terms and pathways were downregulated uniquely after CSW-1 ILTV infection, indicating a viral-strain specific effect in this cell-type. Overall, this study highlights that the observed differences in host and ILTV gene transcription in vitro were more strongly influenced by the cell-types used rather than the presence or absence of gG. This underscores the importance of cell-line selection in studying host-virus interactions and interpreting experimental results.

Is it possible to detect surface antigen CD133 on patient-derived glioblastoma continuous cell cultures using fluorescent aptamers?

To detect CD133 in patient-derived glioblastoma continuous cell cultures using fluorescence microscopy and modified aptamers (molecular recognition elements) anti-CD133. To detect CD133, we used mousey fluorescence monoclonal antibodies anti-CD133 MA1-219, FAM-modified DNA aptamers anti-CD133 AP-1-M and Cs5. Non-aptamer DNA oligonucleotide NADO was used as a negative control. Detection was performed for three samples of patient-derived glioblastoma continuous cell cultures coded as 1548, 1721 and 1793. MA1-219 antibodies brightly stained cell culture 1548, to a lesser extent - 1721. There was diffuse staining of cell culture 1793. Cs5-FAM aptamer stained cells in a similar way, but much weaker. AP-1-M-FAM aptamer interacted with cells even weaker and diffusely stained only cell culture 1793. Non-aptamer NADO did not stain cell culture 1548 and very weakly diffusely stained cell culture 1793. For both molecular recognition elements (MA1-219 antibody and Cs5 aptamer), 3 cell culture samples can be arranged in the following order possibly reflecting CD133 status decrease: strong signal for cell culture 1548, much weaker for 1721, even weaker for 1793. Only cell culture 1548 can be considered CD133 positive with combination of Cs5+ and NADO signals. Cell culture 1793 is CD133 false positive with combination of Cs5+ and NADO+ signals.

Optimization of conditions for cultivation of pathogens of infectious rhinotracheitis and viral diarrhea

This article reflects the results of research on optimizing the conditions of cultivation of strain “ТК-A(VIEV)-В2” of infectious bovine rhinotracheitis (IBR) and strain “ВК-1” of bovine viral diarrhea (BVDV). It was found that the roller cultivation method is most easily applicable and provides the production of viral material with high infectious activity. A comparative analysis of the results of studying the sensitivity of continuous cell cultures to the pathogens of IBR and BVDV of cattle of the latter made it possible to select the MDBK cell line to obtain the maximum titer of viruses. The optimal dose for infecting cell culture with both pathogens was the content of viral particles from 0.2 to 0.5 TCD50/cm3 per cell. The maximum reproduction of viruses was observed using a culture concentration of cells from 110 to 120 thousand cells/cm3 and the duration of their cultivation for 2-3 days. The results obtained can be applied for the further development and creation of preventive and diagnostic tools for IBR and BVDV of cattle.

Biological properties of foot-and-mouth disease virus А 2205/G-IV strain

According to the World Organisation for Animal Health, foot-and-mouth disease (FMD) is regularly reported in domestic and wild cloven-hoofed animals in Africa. G-I, G-IV, G-VI, G-VII, ASIA/Iran-05 genetic lineages of serotype А FMD virus are considered to be the most widespread on the African continent. Given the close economic and trade relations maintained by the Russian Federation with the countries of North Africa, of particular interest for us is studying the FMD virus of serotype A G-IV genetic lineage, which has been responsible for the infection outbreaks in the naturally susceptible animal population of the said region every year since 2012, and there is a risk of introduction of this virus genotype in to the Russian Federation. Therefore, the issues of FMD introduction risk assessment and timely diagnosis are relevant for the Veterinary Service of Russia. FMD virus A 2205/G-IV strain tested for its biological and antigenic properties in cell cultures and naturally susceptible animals (cattle and pigs) was adapted for its reproduction in initially trypsinized porcine kidney (PK) cell culture, continuous monolayer cell cultures (IB-RS-2, PSGK-30, YaDK-04, ВНК-21) by five serial passages. The virus was considered to be adapted when 90–95% cytopathic effect developed within 14–19 hours after the cell culture infection. The virus adapted to the cell cultures was tested for its infectivity with microtitration in IB-RS-2 cell culture. The virus strain tested for vaccine matching with microneutralization test (MNT) demonstrated significant difference from production A/Turkey/06, А22 No. 550/Azerbaijan/64, A22/Iraq/64, A/Iran/97, A No. 2155/Zabaikalsky/2013, A No. 2166/Krasnodarsky/2013, A No. 2269/ARRIAH/2015 strains of FMD virus.

Features of SARS-CoV-2 Replication in Various Types of Reptilian and Fish Cell Cultures.

SARS-CoV-2 can enter the environment from the feces of COVID-19 patients and virus carriers through untreated sewage. The virus has shown the ability to adapt to a wide range of hosts, so the question of the possible involvement of aquafauna and animals of coastal ecosystems in maintaining its circulation remains open. the aim of this work was to study the tropism of SARS-CoV-2 for cells of freshwater fish and reptiles, including those associated with aquatic and coastal ecosystems, and the effect of ambient temperature on this process. In a continuous cell culture FHM (fathead minnow) and diploid fibroblasts CGIB (silver carp), SARS-CoV-2 replication was not maintained at either 25 °C or 29 °C. At 29 °C, the continuous cell culture TH-1 (eastern box turtle) showed high susceptibility to SARS-CoV-2, comparable to Vero E6 (development of virus-induced cytopathic effect (CPE) and an infectious titer of 7.5 ± 0.17 log10 TCID50/mL on day 3 after infection), and primary fibroblasts CNI (Nile crocodile embryo) showed moderate susceptibility (no CPE, infectious titer 4.52 ± 0.14 log10 TCID50/mL on day 5 after infection). At 25 °C, SARS-CoV-2 infection did not develop in TH-1 and CNI. our results show the ability of SARS-CoV-2 to effectively replicate without adaptation in the cells of certain reptile species when the ambient temperature rises.

Adaptation of african swine fever virus (Asfarviridae: Asfivirus)to growth in the continuous culture PPK-66b cells by the method of accelerated passaging

African swine fever virus (ASF) is a large, enveloped virus with an icosahedral capsid morphology and a double-stranded DNA genome ranging in size from 170 to 190 kb. The replication cycle proceeds in two phases, the early phase lasting 4-6 hours and the late 8-20 hours after infection. The adaptation of the ASF virus to growth in continuous cell lines makes efficient and reliable genetic analysis and more accurate interpretation of its results. Adaptation of a new isolate of the ASF virus to growth in a continuous cell line by the method of accelerated passages and preliminary genetic analysis of the resulting strain. For virus isolation and passaging of the ASF virus, a porcine leukocyte cell culture (PL) and continuous cell cultures of porcine origin (ST, PK, PPK-66b) were used with Eagle MEM and HLA essential media with 10% porcine or fetal serum. The article presents data on the isolation and analysis of the changes in the reproductive properties of a new African swine fever (ASF) virus isolate in the process of adaptation to growth in a continuous piglet kidney cell culture clone b (PPK-66b). The current state of the problem of cultivation of the ASF virus, the features of its reproduction, and the basis of the genetic differentiation of its isolates are described in detail. Understanding the uniqueness of the nature of the ASF virus determined the approaches to the processes of its cultivation and adaptation. In this regard, the results of studies of cultural properties, and analysis of the nucleotide sequence of 6 genes of the new isolate, as well as phylogenetic analysis of these genes with already known strains and isolates of the ASF virus are presented. A new strain obtained in the process of cell adaptation of ASVF/Znaury/PPK-23 ASF virus by the accelerated passaging method reaches a high level of reproduction in 72 hours with an accumulation titer of 7.07 lg HAdE50/cm3. Primary genetic analysis allowed to establish the main phylogenetic relationships of the newly isolated strain with previously known variants of the current ASF panzootic.

Senescent cells at the crossroads of aging, disease, and tissue homeostasis.

Originally identified as an outcome of continuous culture of primary cells, cellular senescence has moved beyond the culture dish and is now a bona fide driver of aging and disease in animal models, and growing links to human disease. This cellular stress response consists of a stable proliferative arrest coupled to multiple phenotypic changes. Perhaps the most important of these is the senescence-associated secretory phenotype, or senescence-associated secretory phenotype -a complex and variable collection of secreted molecules release by senescent cells with a number of potent biological activities. Senescent cells appear in multiple age-associated conditions in humans and mice, and interventions that eliminate these cells can prevent or even reverse multiple diseases in mouse models. Here, we review salient aspects of senescent cells in the context of human disease and homeostasis. Senescent cells increase in abundance during several diseases that associated with premature aging. Conversely, senescent cells have a key role in beneficial processes such as development and wound healing, and thus can help maintain tissue homeostasis. Finally, we speculate on mechanisms by which deleterious aspects of senescent cells might be targeted while retaining homeostatic aspects in order to improve age-related outcomes.

Establishment and characterization of a continuous cell culture derived from wood frog (Rana sylvatica) skin

Establishment and characterization of a continuous cell culture derived from wood frog (Rana sylvatica) skin

Dynamics of Ex Vivo Mesenchymal Stromal Cell Potency under Continuous Perfusion.

Mesenchymal stromal cells (MSCs) are a candidate for cell immunotherapy due to potent immunomodulatory activity found in their secretome. Though studies on their secreted substances have been reported, the time dynamics of MSC potency remain unclear. Herein, we report on the dynamics of MSC secretome potency in an ex vivo hollow fiber bioreactor using a continuous perfusion cell culture system that fractionated MSC-secreted factors over time. Time-resolved fractions of MSC-conditioned media were evaluated for potency by incubation with activated immune cells. Three studies were designed to characterize MSC potency under: (1) basal conditions, (2) in situ activation, and (3) pre-licensing. Results indicate that the MSC secretome is most potent in suppressing lymphocyte proliferation during the first 24 h and is further stabilized when MSCs are prelicensed with a cocktail of pro-inflammatory cytokines, IFNγ, TNFα, and IL-1β. The evaluation of temporal cell potency using this integrated bioreactor system can be useful in informing strategies to maximize MSC potency, minimize side effects, and allow greater control for the duration of ex vivo administration approaches.

Microfluidic Gradient Culture Arrays for Cell Pro-oxidation Analysis Using Bipolar Electrochemiluminescence.

A microfluidic gradient array is a widely used screening and analysis device, which has characteristics of high efficiency, high automation, and low consumption. Bipolar electrode electrochemiluminescence (BPE-ECL) has special value in microfluidic array chips. The combination of the microfluidic gradient and BPE arrays has potential for high-throughput screening. In this article, a microfluidic BPE array chip for gradient culture and conditional screening of cancer cells was designed. The generation of concentration gradients, continuous culture of cancer cells with high throughput, and drug screening through BPE-ECL of the Ru(bpy)32+/TPrA system can be performed in one chip. We tested gradient pro-oxidation of MCF-7 by ascorbic acid and the synergistic effect of pro-oxidation on doxorubicin. The method achieves high analysis efficiency through a BPE array while simplifying the tedious procedures required by cell culture methods.

Establishment and characterization of long-term human primary parathyroid tumor subclones derived from Indian PHPT.

The continuous cell line of epithelial human parathyroid cells has been proven difficult. Previously, PTH-C1 cell line was only established rat parathyroid tissue cell line known to express the parathyroid hormone-related peptide (Pthrp) gene. The paucity of continuous cell line of human parathyroid cells secreting parathyroid hormone (PTH) has imposed hurdle in in vitro assessment of the mechanisms involved in the control of parathyroid cell function and proliferation. The primary cell cultures of human parathyroid cells were derived from parathyroid adenoma tissue biopsy (n = 5). The cells were subsequently subcultured to maintained primary subclones. Karyotyping analysis was performed to analyze the genotypic identity of derived subclones. The expression of calcium-sensing receptor (CaSR) and intact parathyroid hormone (iPTH) were analyzed using immunocytochemistry and immunofluorescence. In the present study, we have used a defined condition medium to generate the continuous culture of human parathyroid cells derived from patients with parathyroid adenoma due to primary hyperparathyroidism. The subcultured primary subclones were maintained epithelial and polygonal morphology, doubling time of approximately 25h, displaying a diploid chromosome number, and secretion of PTH. This cell line produces PTH and expresses the calcium-sensing receptor (CaSR) known to be involved in parathyroid function. Altogether these findings indicate the uniqueness of the human parathyroid cell line as an in vitro model for cellular and molecular studies on parathyroid physiopathology.

Abstract 426: Ongoing TCRβ VDJ rearrangement in pre-T LBL cell lines

Abstract The prognosis of recurrent precursor T cell lymphoblastic leukemia/lymphoma (pre-T LBL) is poor, mainly due to chemotherapy resistance. The specific mechanism(s) leading to chemoresistance at relapse is still poorly understood. We previously characterized a mouse strain that contains a CreERT2 cassette “knocked into” the 3′ UTR of Mcm2. Mice with 2 copies of this knock-in allele, designated Mcm2cre/cre show decreases Mcm2 protein expression. They die of pre-T LBL, due to acquired DNA copy number abnormalities (CNA), involving important tumor suppressor or oncogenes. We hypothesize this unique mutator phenotype may reveal specific genetic drivers leading to chemoresistance. We selected six drugs with different mechanisms that are known to be active against pre-T LBL, including cytarabine, etoposide, melphalan, methotrexate, prednisone, and vincristine. We used 4 mcm2 Pre-T LBL cell lines (#2883, 2696, 2641 and 2869). After 1 year of treatment, we obtained resistant cell lines that can tolerate drug concentrations which are up to 10,000X that of parental cell lines. These samples will be characterized by Sparse WGS (for CNA), RNA-Seq and WES. Before sequencing, we wanted to verify that cell lines had not become cross-contaminated during drug selection. During thymocyte differentiation, noncontiguous genomic V, D, and J gene segments become juxtaposed, and form an exon which encodes the variable region of a T cell receptor protein. The TCRβ VDJ rearrangement provides a unique identifier for pre-T LBL cell lines. For the 2869 cell line, we found that the VDJ sequences of cytarabine, vincristine, melphalan, and prednisone resistant 2869 were identical to the parental 2869 cells. However, analysis of 2883 cell lines were more complex. Although there were multiple VDJ rearrangements in parental 2883, they had an identical junction of D2J2S4, suggesting that the variable VDJ rearrangements were derived from a single clone with a unique DJ rearrangement, and subsequent different V-DJ rearrangements. Moreover, following drug selection, the VDJ rearrangement of chemo-resistant 2883 and parental 2883 were not identical. However, the chemo-resistant 2883 cells retain the fingerprint of parental 2883, namely the unique junction of D2J2S4. The VDJ rearrangement of etoposide resistant and vincristine resistant 2883 (2883ER and 2883VR, respectively) clones were identical, and could be identified as a subclone of parental 2883. Interestingly, the2883ER and 2883VR clones had nearly identical CNA of a number of genes, including Abcb1a (also known as Multi-Drug Resistance gene 1a), suggesting selection of a pre-existing resistant clone. Although the VDJ rearrangement of cytarabine 2883 was not found in the parental 2883 subclone, however, the unique D2J2S4 was present. These findings indicate that pre-T LBL cell lines do not invariably have stable TCRβ VDJ rearrangements, and can undergo V-DJ rearrangement event after several years of continuous cell culture. Citation Format: Dengchao Cao, Mianmian Yin, Toshihiro Matsukawa, Taimour Baslan, Peter Aplan. Ongoing TCRβ VDJ rearrangement in pre-T LBL cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 426.

Production of High-Value Proteins under Stringent Cost Constraints—The Case of Hollow Fiber Technology for Cell Culture

For decades, the benefits of utilizing hollow fiber bioreactors for continuous cell culture to produce monoclonal antibodies have been widely recognized. However, the suitability of this technology for laboratories or centers with limited resources and expertise seeking to expand their production capacity is uncertain, mainly due to unknown cost-effectiveness. In this study, a hollow fiber bioreactor with a 4.7-mL culture volume was used to culture a hybridoma clone producing immunoglobulin G antibody specific to hemoglobin F (HbF). The antibody reached a maximum concentration of 1.22 g/L and totaled 21 mg over a 44-day culture period. This preliminary production data was used to estimate the cost of consumables required for using the hollow fiber bioreactor to produce 130 mg of monoclonal antibodies, which was found to be THB35.8k (£880). The cost was slightly more expensive than batch cultivation in typical culture dishes, which ranged from THB27.8 to 30.2k (£680 to 740). Despite the advantages in terms of reduced hands-on time, shorter production duration, and highly concentrated products, the primary challenges associated with using hollow fiber bioreactors were the cost and availability of the cartridges.