Total Viable Cell Research Articles

Development of an 18-color flow cytometry panel for B-lymphoblastic leukemia minimal residual disease detection

Abstract B-cell acute lymphoblastic leukemia (B-ALL) is characterized by a neoplastic proliferation of precursor B cells that have blast cytomorphology and immature immunophenotype. Following treatment of B-ALL, flow cytometry is utilized to detect minimal residual disease (MRD), which aids in monitoring therapeutic response and informing prognosis. In our institution, the BD FACSCanto cytometer is used in conjunction with two standardized 8-antibody EuroFlow panels to detect MRD in B-ALL patients. The objective of this study was to validate a new 18-color B-ALL MRD panel for bone marrow aspirate specimens to be run on a BD LSRFortessa cytometer, with the intention of improving sensitivity and consolidating the testing into one antibody panel. The proposed panel consisted of 22 antibodies, some of which were stacked within the same fluorescence channel, in addition to a viability stain. Twelve bone marrow aspirate specimens collected in 2023 (five positive and seven negative B-ALL MRD specimens) were stained with the new antibody panel and analyzed on the Fortessa cytometer. CD45 dim, CD19-positive blasts were analyzed using BD FACSDiva Software. For the five specimens previously reported as B-ALL MRD-positive using the EuroFlow panels, we were able to detect phenotypically identical B-ALL cells with the 18-color panel. A paired t-test done on the five MRD-positive specimens showed no significant difference in the percentage of residual B-ALL cells (out of total viable cells) measured by the 18-color panel when compared to the EuroFlow panels (EuroFlow/18-color: 0.0096%/0.0097%, 0.024%/0.012%, 0.94%/0.99%, 0.85%/0.85%, 0.022%/0.017%; p=0.58). All seven specimens that were previously reported as B-ALL MRD-negative using the EuroFlow panels were also negative for residual B-ALL cells when stained with the 18-color panel and analyzed on the Fortessa cytometer. The antibody cost per test of the EuroFlow panels ($20-$25, ~$44 for both panels) and new 18-color panel ($63) were calculated based on the unit cost of each antibody aliquot and the required volume of reagent per test. In summary, the new 18-color B-ALL MRD panel run on the BD LSRFortessa cytometer appears to provide similar results as the EuroFlow panels run on the BD FACSCanto cytometer. Although compared to the two EuroFlow panels, the single 18-color panel is more expensive per test in terms of antibodies, it may be preferable when there is scant tissue (such as cerebrospinal fluid) as there may not be enough material to run both EuroFlow panels.

PEITC Induces DNA Damage and Inhibits DNA Repair-Associated Proteins in Human Retinoblastoma Cells In Vitro.

Phenethyl isothiocyanate (PEITC), a natural product, exists in biological activities, including anticancer activity in many human cancer cells. No information shows that PEITC affects DNA damage in human retinoblastoma (RB) cells in vitro. In this study, the aim of experiments was to determine whether PEITC decreased total viable cell number or not by inducing protein expressions involved in DNA damage and repair in Y79 RB cells in vitro. Total cell viability was measured by PI exclusion assay, and PEITC reduced the total Y79 viable cell numbers in a dose-dependent manner. DNA condensation and DNA impairment were conducted by DAPI staining and comet assays, respectively, in Y79 cells. The findings show that PEITC induced DNA condensation dose-dependently based on the brighter fluorescence of cell nuclei stained by DAPI staining. PEITC-induced DNA damage showed a more extended DNA migration smears than that of the control, which was performed by a comet assay. Western blotting was performed to measure the protein expressions involved in DNA damage and repair, which showed that PEITC at 2.5-10 μM increased NRF2, HO-1, SOD (Mn), and catalase; however, it decreased SOD (Cu/Zn) except 10 μM PEITC treatment, and decreased glutathione, which were associated with oxidativestress. Furthermore, PEITC increased DNA-PK, MDC1, H2A.XpSer139, ATMpSer1981, p53, p53pSer15, PARP, HSP70, and HSP90, but decreased TOPIIα, TOPIIβ, and MDM2pSer166 that were associated with DNA damage and repair mechanism in Y79 cells. The examination from confocal laser microscopy shows that PEITC increased H2A.XpSer139 and p53pSer15, and decreased glutathione and TOPIIα in Y79 cells. In conclusion, the cytotoxic effects of PEITC on reducing the number of viable cells may be due to the induction of DNA damage and the alteration of DNA repair proteins in Y79 cells in vitro.

Sources of Variability in Determinations of Brewing Yeast Total Cell Counts and Viability Obtained by Microscopy

Brewers utilizing yeast cell counts and determinations of yeast viability obtained by microscopy require precise results for calculating the yeast pitch to produce fermentations with the highest quality and consistency. The steps in obtaining results with the desired precision include identifying, quantifying and controlling the sources of variation. In this study, we used simulated cell counting, manual cell counting, and semi-automated cell counting to explore sources of variation contributing to the variability of yeast cell counts and yeast viability determinations. The results showed that the variability associated with counting different fields of view contributed most to the variability in yeast cell counts and determinations of yeast viability. Understanding the sources and amounts of variation in cell counts will help brewers direct their efforts toward obtaining the highest quality determinations of yeast cell counts and viability.

Isolating Human Peripheral Blood Mononuclear Cells from Buffy Coats via High Throughput Immunomagnetic Bead Separation.

Peripheral blood mononuclear cells (PBMCs) are a heterogeneous population of monocytes and lymphocytes. Cryopreserved PBMCs have stable viability in long-term storage, making them an ideal cell type for many downstream research purposes, including flow cytometry, immunoassays, and genome sequencing. Typically, PBMCs are isolated viadensity gradient centrifugation, however, it is a low-throughput workflow that is difficult and costly to scale. This article presents a high-throughput workflow using a magnetic bead-based PBMC isolation method that is quick to implement. Total cell concentration, viability, and population distribution with PBMCs obtained using density gradient isolation were compared, and cell viability and proportion of cell types were comparable for both techniques. Isolated PBMCs demonstrated over 70% viability up to 9 days after blood collection, although yield decreased by half after 5 days compared to PBMCs processed within 24 h of collection. In summary, this article describes a PBMC protocol that utilizes a bead-based approach to adapt to a high throughput workflow and demonstrates that both manual and automated bead-based methods can increase processing capacity and provide flexibility for various budgets.

Pemanfaatan Bakteri Asam Laktat (BAL) Amilolitik untuk Fermentasi Tepung Terigu dan Gandum

The national demand for carbohydrates in flour continues to increase significantly due to the emergence of contemporary food products that use flour as the primary ingredient. One of the products used in producing food is sourdough, which is fermented flour. Lactic acid bacteria (LAB) have a role in food fermentation by producing lactic acid. Amylolytic LAB can produce amylase enzyme, a biocatalyst in the starch hydrolysis process. One of the products made from the fermentation of wheat flour is sourdough. This study aims to determine the character of amylolytic LAB in wheat and wheat flour fermentation. The research methods include rejuvenation of LAB, selection of isolates, fermentation of starchy materials, and characterization of fermented liquid. The selection of bacteria was based on cell viability and pH, which showed that isolated Pediococcus pentasaceus E1222 could be used as a starter for starchy fermentation. The highest total acid and cell viability were produced in 24-hour wheat and wheat flour fermentation. Fermented wheat flour and flour had the most favorable aroma and texture, and fermented flour had the most favorable taste. Isolate Pediococcus pentasaceus E1222 is an isolate that can be used in making sourdough.

Development of an Apple Snack Enriched with Probiotic Lacticaseibacillus rhamnosus: Evaluation of the Refractance Window Drying Process on Cell Viability.

The objective of this study was to develop a dried apple snack enriched with probiotics, evaluate its viability using Refractance Window (RWTM) drying, and compare it with conventional hot air drying (CD) and freeze-drying (FD). Apple slices were impregnated with Lacticaseibacillus rhamnosus and dried at 45 °C using RWTM and CD and FD. Total polyphenol content (TPC), color (∆E*), texture, and viable cell count were measured, and samples were stored for 28 days at 4 °C. Vacuum impregnation allowed for a probiotic inoculation of 8.53 log CFU/gdb. Retention values of 6.30, 6.67, and 7.20 log CFU/gdb were observed for CD, RWTM, and FD, respectively; the population in CD, RWTM remained while FD showed a decrease of one order of magnitude during storage. Comparing RWTM with FD, ∆E* was not significantly different (p < 0.05) and RWTM presented lower hardness values and higher crispness than FD, but the RWTM-dried apple slices had the highest TPC retention (41.3%). Microstructural analysis showed that RWTM produced a smoother surface, facilitating uniform moisture diffusion and lower mass transfer resistance. The effective moisture diffusion coefficient was higher in RWTM than in CD, resulting in shorter drying times. As a consequence, RWTM produced dried apple snacks enriched with probiotics, with color and TPC retention comparable to FD.

Exploring the safety and efficacy of GT201 as a first-in-class autologous tumor-infiltrating lymphocyte monotherapy in advanced solid tumors.

2547 Background: GT201, featuring a membrane bound IL-15 stably expressed on tumor-infiltrating lymphocytes (TIL), aims to augment TIL persistence and function as well as ameliorate immune suppression within the tumor microenvironment, offering potential for durable responses in advanced solid tumor patients. We present data from 7 patients enrolled in an open-label, single-arm, multicenter clinical study (NCT05729399), investigating the safety profile and efficiency trend of GT201 therapy. Methods: The GT201 phase 1 trial is designed with the primary endpoint of DLT evaluation and the secondary endpoint of measuring preliminary efficacy parameters including overall response rate (ORR), disease control rate (DCR), progression-free survival (PFS), duration of response (DOR, and overall survival (OS) following the RECIST v1.1. Results: As of January 22, 2024, a cohort of 7 pts has been enrolled in the study, with a median age of 48 and a median of 3 prior lines of therapy. Among these patients, 1 pt has a history of bone metastases, 2 pts have a history of liver metastases, and 1 pt has a history of brain metastases. Following the standard FC lymphodepletion, pts underwent infusion with GT201 at doses ≥ 5x109 total viable cells. Five pts subsequently received IL-2 as part of the treatment protocol. Treatment-related AEs were observed in ≥ 50% of pts with no grade≥3 AEs. Grade ≥ 3 AEs related to FC lymphodepleting chemotherapy and IL-2 included decreased lymphocyte count, decreased neutrophil count, and decreased white blood cell count, pyrexia and increased heart rate. Notably, all grade ≥3 AEs were either recovered or downgraded to grade ≤ 2 within 14 days. Among the 7 response-evaluable patients across various indications, including Non-Small-Cell-Lung Cancer (NSCLC), melanoma malignant, cervical cancer, and ovarian cancer, 3 patients (42.9%) achieved a confirmed partial response (PR), and 2 patients (28.6%) demonstrated stable disease (SD) as their best response. Notably, among the NSCLC subgroup, disease control (SD ≥ 24 weeks or any PR) was observed in all 3 pts (3/3, 100 %). GT201 cells can be detected in all patients receiving treatment, indicated by both staining IL15RA protein on peripheral T cells and measuring the transgene copy number in peripheral white blood cells. GT201 cells expanded robustly in patients and persist in peripheral blood beyond at least 6 months post cell infusion. Conclusions: In patients with heavily pretreated advanced or metastatic solid tumor, GT201, when infused after FC lymphodepletion and followed by high dose IL-2, exhibits a manageable safety profile. Notably, GT201 has demonstrated a favorable clinical profile in NSCLC, with an encouraging objective response rate, response durability, and no GT201-treatment related grade≥3 AEs. Clinical trial information: NCT05729399 .

Application of anthocyanins from blue pea flower in yoghurt and fermented milk: An alternate natural blue colour to spirulina

Studies investigating the application of blue pea flower anthocyanins in fermented dairy products are limited. In this study, the application of anthocyanin extracts from blue pea flower (Clitoria ternatea) in yoghurt and fermented milk at 1 and 2% (w/v) concentrations was investigated and compared to a commercial blue colourant from spirulina. The total anthocyanin content (TAC), antioxidant activity (DPPH free radical and ferric reducing antioxidant power assays), pH, titratable acidity, syneresis, colour, and viable cell count were measured at 6 days intervals for 30 days at 4 °C storage. The TAC of yoghurt with blue pea flower anthocyanin extract demonstrated high stability, but the TAC of fermented milk with blue pea flower anthocyanin extract decreased significantly. The yoghurt and fermented milk with blue pea flower anthocyanin extract demonstrated higher antioxidant activity than the negative control and samples containing spirulina. The total colour change (ΔE) of yoghurt with blue pea flower anthocyanin extract was less than that of yoghurt containing spirulina during storage. However, the colour of fermented milk with blue pea flower anthocyanin extract was less stable than samples containing spirulina. The average colour acceptability of yoghurt and fermented milk containing 2% (w/v) blue pea flower anthocyanin extracts and those containing spirulina colourant was not significantly different. Therefore, an anthocyanin extract from blue pea flowers at 2% (w/v) concentration was a better blue colourant than spirulina in yoghurt. Spirulina was a better colourant compared to blue pea flower anthocyanins for fermented milk.

Point-of-care human milk concentration by passive osmosis: comprehensive analysis of fresh human milk samples.

Preterm infants need enrichment of human milk (HM) for optimal growth. This study evaluated a novel, point-of-care human milk concentration (HMC) process for water removal from fresh HM samples by passive osmotic concentration. Nineteen fresh HM samples were concentrated by incubation with the HMC devices for 3 h at 4 °C. Pre- and post-concentration HM samples were compared by HM properties for: pH, osmolality, macronutrients, enzyme activity, bioactive, and total cell viability. Passive osmotic concentration reduced HM volume by an average of 16.3% ± 3.8% without a significant effect on pH or cell viability. Ten of the 41 HM components did not differ significantly (p > 0.05) between pre- and post-concentration samples. Twenty-three increased within the expected range by volume reduction. Six increased more than expected, two less than expected, and none decreased significantly. Passive osmotic concentration of fresh HM can concentrate HM components by selective removal of water. HM osmolality and pH remained within neonatal feeding parameters.

Effects of dietary supplementation with macroalgae on sperm quality and antioxidant system in Senegalese sole

Within the efforts in the search for novel feed ingredients in aquaculture, macroalgae are being widely explored due to their diversity of bioactive compounds with considerable prophylactic and therapeutic potential. Plocamium cartilagineum and Sargassum vulgare, have the potential to positively impact fish health, mainly due to their antioxidant properties. The production of Senegalese sole (Solea senegalensis) still faces some constraints due to the low sperm quantity and quality of males from captivity, which is highly affected by oxidative stress. We hypothesized that the scavenger ability of P. cartilagineum and S. vulgare could ameliorate the reproductive performance of Senegalese sole males, once incorporated in the broodstock feeds. In this experiment, broodstock groups were fed with three different formulated experimental diets for 60 days: Control (Ctrl), a diet supplemented with 5% P. cartilagineum (Pc), and a diet supplemented with 5% S. vulgare (Sv). Fish were sampled twice to evaluate sperm quality using different techniques: sperm motility by Computer Assisted Sperm Analysis (CASA) software, and cell viability and Reactive Oxygen Species (ROS) by flow cytometer. At the final sampling, 6 fish per treatment were sacrificed and gonads were extracted to determine the relative expression of anti- and pro-apoptotic genes (bcl2b, bcl-x, mcl1b, badb, bax, boka), and genes involved in fish antioxidant system (sod3, cat, gpx, hsp70). Sex steroids were measured on blood plasma by ELISA. Specific growth rate (SGR) and feed efficiency were also evaluated at the end of the experiment. Senegalese sole breeders fed with Pc diet for 60 days showed enhanced sperm total motility and cell viability at the end of the experiment, when compared with the Ctrl diet. On the other hand, fish fed with Sv diet had lower spermatozoa intracellular ROS and upregulated gpx in the gonads. No differences were obtained in levels of sex steroids and gonadal apoptotic system after 60 days of feeding, however, the macroalgae species and the percentages of incorporation used proved to positively modulate different spermatozoa traits.

Isolation and characterization of bio-prospecting gut strains Bacillus safensis CGK192 and Bacillus australimaris CGK221 for plastic (HDPE) degradation.

The present work reports the application of novel gut strains Bacillus safensis CGK192 (Accession No. OM658336) and Bacillus australimaris CGK221 (Accession No. OM658338) in the biological degradation of synthetic polymer i.e., high-density polyethylene (HDPE). The biodegradation assay based on polymer weight loss was conducted under laboratory conditions for a period of 90days along with regular evaluation of bacterial biomass in terms of total protein content and viable cells (CFU/cm2). Notably, both strains achieved significant weight reduction for HDPE films without any physical or chemical pretreatment in comparison to control. Hydrophobicity and biosurfactant characterization were also done in order to assess strains ability to form bacterial biofilm over the polymer surface. The post-degradation characterization of HDPE was also performed to confirm degradation using analytical techniques such as Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), Field emission scanning electronic microscopy (FE-SEM) coupled with energy dispersive X-ray (EDX), and Gas chromatography-mass spectrometry (GC-MS). Interestingly strain CGK221 was found to be more efficient in forming biofilm over polymer surface as indicated by lower half-life (i.e., 0.00032day-1) and higher carbonyl index in comparison to strain CGK192. The findings reflect the ability of our strains to develop biofilm and introduce an oxygenic functional group into the polymer surface, thereby making it more susceptible to degradation.

Ecological Significance of Dynamism of Physiologic Variance on Properties of Palm Oil Mill Effluents

Properties a given waste is essential for rationale comprehension of plausible way(s) for its remediation. In the present study, POME from oil milling centre at Anambra state showed high heterotrophic activity of microorganisms. Total viable counts of the organisms isolated from the waste water were 5.4 x 103 CFU/ml and 4.4 x 103 CFU/ml at day 0 and day 7 of the incubation respectively. These observations showed a significant variation from the control experiment at these recorded days of incubations which shows total viable cell counts (TVC) of 4.6 x 103 and 2.9 x 104 CFU/ml respectively. Total coliform counts (TCC) of the organisms isolated from the dairy waste water were 1.3 x 103 and 1.8 x 103 CFU/ml for the day 0 and day 7 incubation periods respectively while the TCC from the control experiment were: 8.0 x 102 and 3.2 x 102 CFU/ml for day 0 and day 7 incubation periods respectively. POME sample from Awka, Anambra state was subjected to various physicochemical profiling and was further optimized at different conditions to exert various variability. Biochemical oxygen demand (BOD5) is one of the important physical parameter widely used in assessment of quality of aqueous surroundings. Physicochemical properties of waste water from the milling centre showed the following: pH (5.0), temperature (41oC), conductivity (610); water dissolved minerals such as Cl-, K+, PO3, Mg2+, Ca2+ were all determined. Initial dissolved oxygen concentration (mg/l) of the waste water was determined using a probe connected to a meter with initial value of 39.4 mg/l. Biochemical oxygen demand (BOD5) was determined after five incubation days at room temperature in separate bottles optimized at varying pH of 4.0-9.0 in a range of 1.0 units. BOD5 decreases with increased pH values in all the tested samples incubated for 5 days. pH 5.0 gave a significant reduction of the oxygen exertion (1400 mg/ml) while pH 4.0, 6.0, 7.0 showed an oxygen exertion (BOD5) of 1978, 2778, and 2965 mg/ml respectively. Samples incubated at 8.0 and 9.0 showed oxygen exertion of 2945 and 2899 mg/ml respectively. Other contents of the dairy waste water such as total organic carbon (TOC), total organic matter contents (TOM) were all determined respectively. Samples was also collected from mapped area 0.5 km away from the waste water reservoir and used as the reference standard. Proceeding results from the present research study showed the compromised statues of our aquatic environment and also in a wider look gave a great insight of effective modalities in water treatment; as increasing population of myriads of processing industries in our country today pose great challenge to the competence of our aquatic body.

Assessment of immune cells in the uterine fluid at the time of the embryo transfer.

Although endometrial receptivity is a key factor in influencing implantation in both naturally conceived and assisted reproductive technology (ART) cycles, very little is known about the endometrium milieu around the time of implantation. Previous studies have demonstrated the presence of several cytokines in the endometrium that affect implantation. However, there is lacking data about the presence of immune cell subtypes within the endometrium and in the uterine cavity at the time of implantation. This study was approved by the Institutional Review Board (# 225589). The study was designed as a prospective observational cohort study between May 2021 and December 2022 at a single academic-based fertility center. All patients underwent at least one In Vitro Fertilization (IVF) cycle and have frozen embryos. Twenty-four participants were recruited for this study which was conducted during the frozen embryo transfer (FET) cycle regardless of the outcome of previous cycles. Two samples were acquired from each subject, denoted as lower and upper. A trial transfer catheter was introduced under ultrasound guidance into the lower uterine segment. Upon removal, the tip was rinsed in IMDM medium containing 10% FBS (lower uterus). A transfer catheter was then loaded with the embryo that was placed in the upper uterus under ultrasound guidance. The tip of the transfer catheter was rinsed in separate aliquot of the above media (upper uterus). After centrifugation, pelleted cells were stained for the following surface markers: CD45, CD3, CD19, CD4, CD8, gamma delta TCR, CD25, CD127, CD66b, CD14, CD16, CD56 and acquired on Sony SP6800 Spectral Analyzer. Upon staining the pelleted cells, we were able to identify viable leukocytes from samples obtained from both, upper and lower uterus (0.125×106 cells ± SD 0.32), (0.123×106 cells ± SD 0.12), respectively. Among total viable cells, there was no significant difference in both percent and number of CD45+ cells between the upper and lower uterus (9.88% ± 6.98 SD, 13.67% ± 9.79 SD, p=.198) respectively. However, there was significantly higher expression of CD3+ (p=.006), CD19+ (p=.032) and CD14+ (p=.019) cells in samples collected from upper compared to lower uterus. Within all CD3+ cells, we found that gamma delta T cells (GDT) were the major population of T cells in both upper and lower uterus. In contrast, CD8+ T cells were significantly higher in the lower uterus when compared to the upper uterus (p=.009). There was no statistically significant difference in the expression of CD4+ T cells, T regulatory cells (CD4+CD25+CD127-), NK cells (CD56+), neutrophils (CD66b+) and FcγRIII+ cells (CD16+) between upper and lower uterus. We believe the immune milieu at the time of embryo transfer will affect implantation. Understanding the composition of immune cells will guide further research in identifying optimal immune milieus that favor implantation. Comprehensive analysis of endometrium is expected to lead to new diagnostic and therapeutic approaches to improve IVF outcomes.

Exposure to microplastics renders immunity of the thick-shell mussel more vulnerable to diarrhetic shellfish toxin-producing harmful algae

Despite both microplastics (MPs) and harmful algae blooms (HABs) may pose a severe threat to the immunity of marine bivalves, the toxification mechanism underlying is far from being fully understood. In addition, owing to the prevalence and sudden occurrence characteristics of MPs and HABs, respectively, bivalves with MP-exposure experience may face acute challenge of harmful algae under realistic scenarios. However, little is known about the impacts and underlying mechanisms of MP-exposure experience on the susceptibility of immunity to HABs in bivalve mollusks. Taking polystyrene MPs and diarrhetic shellfish toxin-producing Prorocentrum lima as representatives, the impacts of MP-exposure on immunity vulnerability to HABs were investigated in the thick-shell mussel, Mytilus coruscus. Our results revealed evident immunotoxicity of MPs and P. lima to the mussel, as evidenced by significantly impaired total count, phagocytic activity, and cell viability of haemocytes, which may result from the induction of oxidative stress, aggravation of haemocyte apoptosis, and shortage in cellular energy supply. Moreover, marked disruptions of immunity, antioxidant system, apoptosis regulation, and metabolism upon MPs and P. lima exposure were illustrated by gene expression and comparative metabolomic analyses. Furthermore, the mussels that experienced MP-exposure were shown to be more vulnerable to P. lima, indicated by greater degree of deleterious effects on abovementioned parameters detected. In general, our findings emphasize the threat of MPs and HABs to bivalve species, which deserves close attention and more investigation.

Abstract 636: Coculture model for investigating cellular interactions between colorectal tumor organoids and cancer-associated fibroblasts

Abstract The tumor microenvironment (TME) is well recognized for its role in tumor progression, chemoresistance, and recurrence. The TME consists of various components, such as extracellular matrix (ECM), chemical and mechanical cues, as well as stromal cells and their interactions. As the most abundant cells in tumor stroma, cancer-associated fibroblasts (CAFs) have drawn a lot of attention. It has been reported that CAFs not only directly interact with cancer cells via surface proteins and cytokines, but also influence their behaviors by remodeling the ECM. However, due to their heterogenous characteristics and diverse functions, the influence of tumor-CAF interactions on drug response is not fully understood. Here, we present a coculture model for investigating CAF effects on patient-derived colorectal tumor organoids (CTOs) growth, differentiation, and drug response. It consists of normal colon fibroblasts, CCD18-Co, or patient-derived CAFs seeded with CTOs in a mixed hydrogel containing 90% basement membrane extract (BME) and 10% collagen type I. Media conditions were optimized (1:1 mix of CTO culture media plus reduced media excluding growth factors and inhibitors) to achieve similar organoid sizes in the monoculture and coculture conditions at the onset of drug testing. To support higher throughput investigations, our workflow incorporates a liquid handler for automated CTO seeding, media and drug exchange, and an automated microscope with a robotic arm for multi-plate imaging. Briefly, single cell CTOs and fibroblasts were seeded at a 1:1 ratio (3000 total cells/well) in 96-well plates. After 4 days in culture, anti-cancer drugs were administrated in reduced media to the samples. Brightfield images were acquired on days 0, 3, and 5 after drug administration. The collected images were analyzed with a neural network (NN) to dynamically monitor the growth and viability of individual organoids. The CellTiter-Glo assay was performed on day 5 to quantify the total CTO cell viability. The normalized drug response (NDR) was calculated from the NN processed images to determine the drug response. An immunostaining panel, which includes protein markers such as CK20, Ki67, α-SMA, Vimentin, and FAP, was used to evaluate the fibroblast phenotypes and CTO differentiation. Additionally, the cultures were subjected to fluorescent lifetime imaging microscopy (FLIM), a label-free imaging technique, to measure the metabolic adaptations of each cell population. Preliminary studies revealed a CAF-dependent increase in CTO growth and glycolytic signatures in the co-culture setting; however, minimal effects on drug response to common chemotherapy agents such as 5-fluorouracil and irinotecan were observed. In the future, we will use this integrated pipeline as a screening method to identify new treatment strategies aimed at targeting the stromal microenvironment in colorectal cancer. Citation Format: Yuyuan Zhou, Yu-Kai Huang, Emma J. Fong, Elizabeth Elton, Erin McManus, Brandon Choi, Seungil Kim, Shannon M. Mumenthaler. Coculture model for investigating cellular interactions between colorectal tumor organoids and cancer-associated fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 636.

Abstract 2125: The use of intestinal organoids as a preclinical screen to assess gastrointestinal (GI) toxicity and barrier integrity

Abstract Gastrointestinal (GI) toxicity is a common and often severe limiting factor in the development of antineoplastic drugs. Symptoms include diarrhoea, dehydration and ulceration which increase susceptibility to infection, partly due to damage of crypt and/or villus structures in the small intestine, impairing the barrier function. As novel targeted therapies are emerging, assessment of their potential GI toxicity remains crucial. Small intestinal (SI) organoids are 3D in vitro models of the epithelium which recapitulate the structure and function of the small intestine. We have further developed and validated the organoid model as a screening tool to predict GI toxicity and subsequent mucosal regeneration in four species: mouse, rat, dog and human. The culture conditions have been designed to mimic the stem cell niche and allow cell differentiation and proliferation to occur. All intestinal lineages were present in the organoids derived from each species and the epithelial hierarchy closely resembled that observed in vivo. The toxic effects of antineoplastic drugs on the organoids were assessed by quantification of the total cell viability upon treatment, coupled to morphometric analysis of the organoids (size, area, perimeter, and branching efficiency). Immunofluorescence and RNAseq can be used to identify the targeted cells and the toxicity mechanisms of the drugs. To further assess the effect of drugs on barrier integrity, we have developed transwell-grown cell monolayers derived from normal human small intestinal organoids. This model allows direct access to both apical and basal surfaces, which is not possible when using organoids where their geometry prevents access to the apical surface. The monolayer formation was followed by live imaging and Trans-Epithelial Electrical Resistance (TEER) measurements and treatments with toxic drugs were applied after TEER reached a plateau. The direct effect on barrier integrity was evaluated by TEER reduction, increased FITC-Dextran permeability and analysis of TJ proteins by immunofluorescence. Furthermore, effects on cell death were assessed by measuring the levels of Lactate Dehydrogenase (LDH) released into the medium by dying cells. In conclusion, our models provide an innovative in vitro solution to further study the toxic effects of drugs on the normal small intestine and to analyse direct effects on barrier integrity. The monolayer assay may also be useful for developing models of transient TJ impairment to improve the bioavailability of orally administered antineoplastic drugs. The model can be considered more representative of the normal intestine than transformed cell lines such as CaCo2. Citation Format: Valentina Ubertini, Sarah Hall, Frida Ponthan, James Wilson. The use of intestinal organoids as a preclinical screen to assess gastrointestinal (GI) toxicity and barrier integrity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2125.

Application of anthocyanins from black goji berry in fermented dairy model food systems: An alternate natural purple color

The application of anthocyanin extracts from black goji berry (Lycium ruthenicum Murray) in yoghurt and fermented milk at 1 and 2 g/100 mL concentrations was investigated and compared to a commercial colorant, purple sweet potato. The total anthocyanin content, antioxidant activity, pH, titratable acidity, syneresis, color, and viable cell count were measured during storage at 4 °C. The total anthocyanin content of yoghurt with black goji berry anthocyanin extract demonstrated high stability during storage. The yoghurt and fermented milk with black goji berry anthocyanin extract demonstrated higher antioxidant activity than the negative control and samples containing purple sweet potato colorant. The total color change of the yoghurt and fermented milk with black goji berry anthocyanin extract was less than samples containing purple sweet potato colorant during storage. All samples retained a viable cell count of more than 7 log CFU/g at the end of storage. The average color acceptability of yoghurt and fermented milk containing black goji berry anthocyanin extract did not significantly differ from those containing purple sweet potato colorant. Therefore, an anthocyanin extract from black goji berry at 2 g/100 mL concentration was a better natural purple colorant than purple sweet potato in yoghurt and fermented milk systems.

Metabolic engineering of Bcat1, Adh5 and Hahdb towards controlling metabolic inhibitors and improving performance in CHO cell-cultures

CHO cells are known to secrete growth inhibitory metabolites during growth and production phases, which hampers cellular performance and negatively impacts final productivity and product quality attributes. Previous studies have identified different metabolic by-products derived from CHO metabolism and demonstrated their negative impacts on growth and titer productivity. This work presents a control strategy that incorporated genetic engineering and inhibitory metabolites pathway analysis to regulate cellular metabolism. In this study, three different metabolic genes involving the metabolism of branched-chain amino acids (Adh5, Bcat1 and Hadhb) were cloned from CHO reversely synthesized cDNA to study for gene functionality towards regulating cellular metabolism. Identified metabolic genes were individually engineered into expression vector and transfected to cells at plasmid concentration of 40 µg/µL. For all engineered conditions, the peak VCD profile of cells on Day 4 realized a 18–20% increase with cumulative VCD profile realizing 16–19% increase in terms of total viable cells collected on harvest day. Increase in IgG1 titer production and also more complex glycosylation profiles formation were also observed from all engineered subclones. This study hereby successfully demonstrated rewiring cellular metabolism through up-regulation of key metabolic enzymes can effectively control the accumulation of process inhibitors which therefore allowed improvement desirable critical process attributes.

Efficacy of ultrasonic cavitation in isolation of the stromal vascular fraction from adipose tissue

Background Adipose tissue-derived stem cells (ASCs) represent a rapidly evolving area of cell-based therapies. ASCs are harvested from the stromal vascular fraction (SVF), a heterogeneous mixture of ASCs and other cell types. Two methods are typically employed to obtain the SVF: enzymatic and mechanical. In this study, we examined the efficacy of ultrasonic cavitation in isolating the SVF from adipose tissue.Methods Human adipose tissue was procured from three patients through an aesthetic liposuction procedure. This tissue was then subjected to either an enzymatic method utilizing type II collagenase or an ultrasonic cavitation method using an Ultra Stemcell device. The cell counts and viability were determined using a cell counter. The immunophenotype of the SVF was analyzed using real-time polymerase chain reaction.Results Compared to the enzymatic method, the total cell count and cell viability of the SVF isolated through ultrasonic cavitation were relatively low. However, no significant difference was observed in the immunophenotype of CD45, CD11b, CD34, and CD105.Conclusions The ultrasonic cavitation method constitutes a clinically practical approach that offers straightforward access in the operative field. This technique is a rapid and cost-effective method for isolating the SVF.

NMR and multivariate methods: Identification of chemical markers in extracts of pedra-ume-caá and their antiglycation, antioxidant, and enzymatic inhibition activities.

In Brazil, the plant group popularly known as "pedra-ume-caá" is used in folk medicine for the treatment of diabetes, and its raw material is commonly sold. The aim of the study was to apply a method for chemical identification of extracts of dry pedra-ume-caá leaves using HPLC-high-resolution mass spectrometry (HRMS) and NMR and develop a multivariate model with NMR data to authenticate commercial samples. In addition, to evaluate the biological activities of the extracts. Dry extracts of Myrcia multiflora, Myrcia amazonica, Myrcia guianensis, Myrcia sylvatica, Eugenia punicifolia leaves, and 15 commercial samples (sold in Manaus and Belém, Brazil) were prepared by infusion. All the extracts were analysed using HPLC-high-resolution mass spectrometry (HRMS), NMR, principal component analysis (PCA), and hierarchical cluster analysis (HCA). The antidiabetic effect of extracts was evaluated according to enzymatic inhibition. Their content of total phenols, cell viability, and antioxidant and antiglycation activities were also determined. HPLC-HRMS and NMR analysis of these extracts permitted the identification of 17 compounds. 1H NMR data combined with multivariate analyses allowed us to conclude that catechin, myricitrin, quercitrin, and gallic and quinic acids are the main chemical markers of pedra-ume-caá species. These markers were identified in 15 commercial samples of pedra-ume-caá. Additionally, only the extracts of M. multiflora and E. punicifolia inhibited α-glucosidase. All the extracts inhibited the formation of advanced glycation end products (AGEs) and showed free-radical-scavenging activity. These extracts did not present cytotoxicity. This study revealed the chemical markers of matrices, and it was possible to differentiate the materials marketed as pedra-ume-caá. Moreover, this study corroborates the potential of these species for treating diabetes.