Impact of baseline culture conditions of mouse-derived cancer organoids when determining therapeutic response and tumor heterogeneity

Representative models are needed to screen new therapies for patients with cancer. Cancer organoids are a leap forward as a culture model that faithfully represents the disease. Mouse-derived cancer organoids (MDCOs) are becoming increasingly popular, however there has yet to be a standardized method to assess therapeutic response and identify subpopulation heterogeneity. There are multiple factors unique to organoid culture that could affect how therapeutic response and MDCO heterogeneity are assessed. Here we describe an analysis of nearly 3,500 individual MDCOs where individual organoid morphologic tracking was performed. Change in MDCO diameter was assessed in the presence of control media or targeted therapies. Individual organoid tracking was identified to be more sensitive to treatment response than well-level assessment. The impact of different generations of mice of the same genotype, different regions of the colon, and organoid specific characteristics including baseline size, passage number, plating density, and location within the matrix were examined. Only the starting size of the MDCO altered the subsequent growth. Here we establish organoid culture parameters for individual organoid morphologic tracking to determine therapeutic response and growth/response heterogeneity for translational studies using murine colorectal cancer organoids.

Microcurrent stimulates cell proliferation and modulates cytokine release in fibroblast cells

Aims: To analyse the effects of microcurrent on L929 fibroblast cell culture. Methods: Cells were cultivated in six-well plates at densities of 5×104, 1×105, 3×105 and 5×105 cells/well to determine the best plating density. Subsequently, two methods of current application were tested: with a paper cone coupled to the electrode (M1) and with the electrode directly inside the well (M2). Then, streams of 60µA (G60), 100µA (G100), 500µA (G500) and 900µA (G900) were applied to the cells (n=3) once a day for three minutes, for a period of one (T1), two (T2) and three days (T3). The MTT assay method was used to evaluate cell proliferation. For the quantification of the inflammatory markers by flow cytometry, the group and time that presented the best results were selected. Results: The ideal plating density was established as 1x105 cells/well and M2 as the best application method. An increase in cell viability was observed at all intensities from T1 to T2, but with no significant differences. From T2 to T3, there was a decrease in viability in all groups, with a significant difference only in G500 (p<0.05). Flow cytometry was performed in the GC and G900 groups at T2. It was possible to observe an increase of 0.56pg/ml in Interleukin (IL)-17 and a decrease of 5.45pg/ml in IL-2. Conclusion: This study showed that two applications of microcurrent increases cell proliferation and modulates the inflammatory response, aiding tissue regeneration and playing a key role in rehabilitation.

Challenges in corneal endothelial cell culture

Corneal endothelial cells (CECs) facilitate the function of maintaining the transparency of the cornea. Damage or dysfunction of CECs can lead to blindness, and the primary treatment is corneal transplantation. However, the shortage of cornea donors is a significant problem worldwide. Thus, cultured CEC therapy has been proposed and found to be a promising approach to overcome the lack of tissue supply. Unfortunately, CECs in humans rarely proliferate in vivo and, therefore, can be extremely challenging to culture in vitro. Several promising cell isolation and culture techniques have been proposed. Multiple factors affecting the success of cell expansion including donor characteristics, preservation and isolation methods, plating density, media preparation, trans-differentiation and biomarkers have been evaluated. However, there is no consensus on standard technique for CEC culture. This review aimed to determine the challenges and investigate potential options that would facilitate the standardization of CEC culture for research and therapeutic application.

Differentiation and Maturation Effect of All-trans Retinoic Acid on Cultured Fetal RPE and Stem Cell-Derived RPE Cells for Cell-Based Therapy

Background: Phase I/II clinical trials using fetal retinal pigment epithelium (fRPE), human embryonic stem cell (hESC)-derived RPE, or human induced pluripotent stem cell (hiPSC)-derived RPE as potential sources of materials for cell-based therapy to treat degenerative retinal diseases have been carried out during the past decade. Challenges for successful translational cell-based therapy include cell manufacture, cell quality, cell storage, and cell behavior in vivo. In this study, we investigated the culture-induced changes in passaged fetal RPE, hESC-RPE and hiPSC-RPE cells in vitro and explored the differentiation and maturation effect of all-trans retinoic acid (ATRA) on those RPE cells. Methods: A total of 9 fetal RPE cell lines, hESC-RPE and hiPSC-RPE cell lines were set up using previously described methods. The culture-induced changes in subsequent passages caused by manipulating plating density, dissociation method and repeated passaging were studied by microscope, real-time quantitative PCR, western blot and immunofluorescent assays. Gene and protein expression and functional characteristics of fRPE, hESC-RPE and hiPSC-RPE incubated with ATRA at different concentration were also evaluated.Results: Compared with fRPE, hESC-RPE and hiPSC-RPE showed decreased gene and protein expression of RPE markers. Passage 3 RPE of all three types seeded at a density of 6×105 and 9x105 cells/mL in basal medium maintained pigmented polygonal, cobblestone-like morphology. RPE cells underwent mesenchymal changes showing increased expression of mesenchymal markers including a-SMA, N-cadherin, fibronectin and decreased expression of RPE markers including RPE65, E-cadherin and ZO-1, as a subsequence of low plating density, inappropriate dissociated method, and repeated passaging. fRPE, hESC-RPE and iPSC-RPE treated by ATRA at different concentrations showed increased expression of RPE markers such as RPE65, bestrophin (BEST) and CRALBP, and increased expression of negative complement regulatory proteins (CRP) including complement factor H (CFH), CD46, CD55 and CD59, and increased transepithelial resistance (TER) as well.Conclusion: Although hESC and hiPSC-derived RPE are morphologically similar to fRPE, and also have the tendency to undergo epithelial-to-mesenchymal transition (EMT) changes during the culturing and passaging process in vitro, differences in protein and gene expression among three RPE types exist. Moreover, ATRA can increase RPE markers expression, as well as to increase the expression levels of CRPs gene and protein in fRPE and stem cell-derived RPE.

Facilitation of mouse skin-derived precursor growth and yield by optimizing plating density

Multiple methodologies have been reported to facilitate skin-derived precursor (SKP) growth, but the impact of plating density on SKP growth has not been studied. To determine the optimal plating density, we used six plating densities and two types of flasks for mouse SKP (mSKP) culture. On the 14th day, the number, diameter, and viability of mSKP spheres were compared by morphological assessment and cell counting kit 8, and we found the optimal plating density was 2.5 × 105–5 × 105 cells/mL. In addition, we investigated the correlation between the SKP spheres and the adherent cell colonies in the serum-free culture system. We treated the adherent cell colonies with two culture conditions and characterized the cells generated from two conditions by immunocytochemistry and induced differentiation, respectively. The results elucidated that the adherent cell colonies differentiated into either mSKPs or dermal mesenchymal stem cells under appropriate culture conditions. In conclusion, mSKP spheres differentiated from the adherent cell colonies. The optimal plating density significantly promoted and advanced the proliferation of adherent cell colonies, which optimized mSKP growth and yield. The adherent cell colonies possessed the capacity of differentiating into different types of cells under appropriate culture conditions.

Differentiation and Maturation Effect of All-trans Retinoic Acid on Cultured Fetal RPE and Stem Cell-Derived RPE Cells for Cell-Based Therapy

Background: Phase I/II clinical trials using fetal retinal pigment epithelium (fRPE), human embryonic stem cell (hESC)-derived RPE, or human induced pluripotent stem cell (hiPSC)-derived RPE as potential sources of materials for cell-based therapy to treat degenerative retinal diseases have been carried out during the past decade. Challenges for successful translational cell-based therapy include cell manufacture, cell quality, cell storage, and cell behavior in vivo. In this study, we investigated the culture-induced changes in passaged fetal RPE, hESC-RPE and hiPSC-RPE cells in vitro and explored the differentiation and maturation effect of all-trans retinoic acid (ATRA) on those RPE cells. Methods: A total of 9 fetal RPE cell lines, hESC-RPE and hiPSC-RPE cell lines were set up using previously described methods. The culture-induced changes in subsequent passages caused by manipulating plating density, dissociation method and repeated passaging were studied by microscope, real-time quantitative PCR, western blot and immunofluorescent assays. Gene and protein expression and functional characteristics of fRPE, hESC-RPE and hiPSC-RPE incubated with ATRA at different concentration were also evaluated.Results: Compared with fRPE, hESC-RPE and hiPSC-RPE showed decreased gene and protein expression of RPE markers. P3 RPE of all three types seeded at a density of 6×105 and 9x105 cells/mL in basal medium maintained pigmented polygonal, cobblestone-like morphology. RPE cells underwent mesenchymal changes showing increased expression of mesenchymal markers including a-SMA, N-cadherin, fibronectin and decreased expression of RPE markers including RPE65, E-cadherin and ZO-1, as a subsequence of low plating density, inappropriate dissociated method, and repeated passaging. fRPE, hESC-RPE and iPSC-RPE treated by ATRA at different concentrations showed increased expression of RPE markers such as RPE65, bestrophin (BEST) and CRALBP, and increased expression of negative complement regulatory proteins (CRP) including complement factor H (CFH), CD46, CD55 and CD59, and increased transepithelial resistance (TER) as well.Conclusion: Although hESC and hiPSC-derived RPE are morphologically similar to fRPE, and also have the tendency to undergo epithelial-to-mesenchymal transition (EMT) changes during the culturing and passaging process in vitro, differences in protein and gene expression among three RPE types exist. Moreover, ATRA can increase RPE markers expression, as well as to increase the expression levels of CRPs gene and protein in fRPE and stem cell-derived RPE.

Organoid model of urothelial cancer: establishment and applications for bladder cancer research

3D cancer cell models are suitable for drug evaluation because they more precisely mimic tissue architecture than 2D cultures. To study cytotoxicity of anticancer agents, the most sensitive CellTiter-Glo 3D assay is used. However, this is an end point assay, so it is not possible to consider the variance of the starting material amount in the final reading. It is difficult to maintain an even plating density of 3D organoids for cytotoxicity analysis. We present a simple, 3D bladder cancer culture that can be maintained, cryopreserved and used for molecular and drug response studies. We applied a simple modification of the drug response assay for 3D cultures by measuring the background signal with the CellTiter Blue assay before drug application.

Protoplast Isolation and Shoot Regeneration from Protoplast-Derived Callus of Petunia hybrida Cv. Mirage Rose

Despite the increasing use of protoplasts in plant biotechnology research, shoot regeneration from protoplasts remains challenging. In this study, we investigated the factors involved in protoplast isolation, callus induction, and shoot regeneration in Petunia hybrida cv. Mirage Rose. The following conditions were found to be most optimal for protoplast yield and viability: 0.6 M mannitol, 2.0% cellulase, and 6 h digestion time. A plating density of 10 × 104 protoplasts/mL under osmoticum condition (0.58 M mannitol) showed high microcolony viability in liquid culture. The Kao and Michayluk medium was found to be appropriate for callus proliferation from microcalli under a 16-h light photoperiod. Calli cultured in Murashige and Skoog medium containing 1.0 mg/L 6-benzylaminopurine and 0.2 mg/L 3-indole butyric acid showed the highest shoot regeneration frequency and number of shoots obtained per explant. Random amplification of polymorphic DNA analysis showed that the protoplast-derived shoots exhibited the same banding patterns as those of donor plants. Collectively, these findings can contribute to solving problems encountered in protoplast isolation and shoot regeneration in other petunia cultivars and related species. As the protocol developed by us is highly reproducible, it can be applied in biotechnology research on P. hybrida cv. Mirage Rose.

Culture of IMR90 Cells

This protocol describes the thawing, culturing, and cryopreservation of the human lung fibroblast cell line IMR90. The attached methods document is a formal version of the information included here. The attached worksheet is a fillable PDF that can be used to maintain cell passage records using this protocol. Please note: Deviation from the three-day passage cycle and cell plating density described here typically results in greater culture and experimental variability.Disclaimer: The contents of this article have been reviewed by the US Environmental Protection Agency and approved for publication and do not necessarily represent Agency policy. Mention of trade names or commercial products does not constitute endorsement or recommendations for use.

Culture of 16HBE14o- Cells

This protocol describes the thawing, culturing, and cryopreservation of the human bronchial epithelial cell line 16HBE14o- (referred to as “16HBE”). The attached methods document is a formal version of the information included here. The attached worksheet is a fillable PDF that can be used to maintain cell passage records using this protocol.Please note: Deviation from the three-day passage cycle and cell plating density described here typically results in greater culture and experimental variability.Disclaimer: The contents of this article have been reviewed by the US Environmental Protection Agency and approved for publication and do not necessarily represent Agency policy. Mention of trade names or commercial products does not constitute endorsement or recommendations for use.