Intestinal epithelial cells derived from human pluripotent stem cells (hPSCs) are generally maintained and cultured as organoids in vitro because they do not exhibit adhesion when cultured. However, the three-dimensional structure of organoids makes their use in regenerative medicine and drug discovery difficult. Mesenchymal stromal cells are found near intestinal stem cells in vivo and provide trophic factors to regulate stem cell maintenance and proliferation, such as BMP inhibitors, WNT, and R-spondin. In this study, we aimed to use mesenchymal stromal cells isolated from hPSC-derived intestinal organoids to establish an in vitro culture system that enables stable proliferation and maintenance of hPSC-derived intestinal epithelial cells in adhesion culture.
We established an isolation protocol for intestinal epithelial cells and mesenchymal stromal cells from hPSCs-derived intestinal organoids and a co-culture system for these cells. We then evaluated the intestinal epithelial cells and mesenchymal stromal cells\' morphology, proliferative capacity, chromosomal stability, tumorigenicity, and gene expression profiles. We also evaluated the usefulness of the cells for pharmacokinetic and toxicity studies.
The proliferating intestinal epithelial cells exhibited a columnar form, microvilli and glycocalyx formation, cell polarity, and expression of drug-metabolizing enzymes and transporters. The intestinal epithelial cells also showed barrier function, transporter activity, and drug-metabolizing capacity. Notably, small intestinal epithelial stem cells cannot be cultured in adherent culture without mesenchymal stromal cells and cannot replaced by other feeder cells. Organoid-derived mesenchymal stromal cells resemble the trophocytes essential for maintaining small intestinal epithelial stem cells and play a crucial role in adherent culture.
The high proliferative expansion, productivity, and functionality of hPSC-derived intestinal epithelial cells may have potential applications in pharmacokinetic and toxicity studies and regenerative medicine.

Single-cell functional analysis provides a natural next step in the now widely adopted single-cell mRNA sequencing studies. Functional studies can be designed to study cellular context by using single-cell culture, perturbation, manipulation, or treatment. Here we present a method for a functional study of 48 single cells by single-cell isolation, dosing, and mRNA sequencing with an integrated fluidic circuit (IFC) on the Fluidigm® Polaris™ system. The major procedures required to execute this protocol are (1) cell preparation and staining; (2) priming, single-cell selection, cell dosing, cell staining, and cDNA generation on the Polaris IFC; and (3) preparation and sequencing of single-cell mRNA-seq libraries. The cell preparation and staining steps employ the use of a universal tracking dye to trace all cells that enter the IFC, while additional fluorescence dyes chosen by the user can be used to differentiate cell types in the overall mix. The steps on the Polaris IFC follow standard protocols, which are also described in the Fluidigm user documentation. The library preparation step adds Illumina® Nextera® XT indexes to the cDNA generated on the Polaris IFC. The resulting sequencing libraries can be sequenced on any Illumina sequencing platform.

Adherent culture which is used to collect adipose tissue extract (ATE) previously brings the problem of inhomogeneity and non-repeatability. Here we aim to extract ATE with stirred suspension culture to speed up the extraction process, stabilize the yield, and improve consistent potency metrics of ATE.
ATE was collected with adherent culture (ATE-A) and stirred suspension culture (ATE-S) separately. Protein yield and composition were detected by SDS-PAGE, while cytokines in ATE were determined with ELISA. The adipogenic and angiogenic potential of ATE were compared by western blot and qPCR. In addition, haematoxylin and eosin staining and lactate dehydrogenase (LDH) activity assays were used to analyze the cell viability of adipose tissue cultured with different methods.
The yield of ATE-S was consistent while ATE-A varied notably. Characterization of the protein composition and exosome-like vesicles (ELVs) indicated no significant difference between ATE-S and ATE-A. The concentrations of cytokines (VEGF, bFGF, and IL-6) showed no significant difference, while IGF in ATE-S was higher than that in ATE-A. ATE-S showed upregulated adipogenic and angiogenic potential compared to ATE-A. Morever, stirred suspension culture decreased the LDH activity of ATE while increased the number of viable adipocytes and reduced adipose tissue necrosis.
Compared with adherent culture, stirred suspension culture is a reliable, time- and labor-saving method to collect ATE, which might be used to improve the downstream applications of ATE.

This chapter describes a method for isolation, maintenance, and propagation of primary glioblastoma (GBM) cells in adherent monolayer cultures from patient tumor specimens. This method enables the establishment of GBM cultures with stem or progenitor-like cell characteristics, including self-renewal capacity, differentiation along restricted neural lineages, and tumor-initiating potential when orthotopically injected into immunocompromised mice. This experimentally tractable model system is therefore suitable for a wide variety of analyses in vitro as well as in vivo. Key examples of biological analyses that can be performed using these cells are also described.

While large-scale culture of insect cells will need to be conducted using bioreactors up to 10,000 l scale, many of the main challenges for cell culture-based production of insecticidal viruses can be studied using small-scale (20-500 ml) shaker/spinner flasks, either in free suspension or using microcarrier-based systems. These challenges still relate to the development of appropriate cell lines, stability of virus strains in culture, enhancing virus yields per cell, and the development of serum-free media and feeds for the desired production systems. Hence this chapter presents mainly the methods required to work with and analyze effectively insect cell systems using small-scale cultures. Outlined are procedures for quantifying cells and virus and for establishing frozen cells and virus stocks. The approach for maintaining cell cultures and the multiplicity of infection (MOI) and time of infection (TOI) parameters that should be considered for conducting infections are discussed.The methods described relate, in particular, to the suspension culture of Helicoverpa zea and Spodoptera frugiperda cell lines to produce the baculoviruses Helicoverpa armigera nucleopolyhedrovirus, HearNPV, and Anticarsia gemmatalis multicapsid nucleopolyhedrovirus, AgMNPV, respectively, and the production of the nonoccluded Oryctes nudivirus, OrNV, using an adherent coleopteran cell line.

Mesenchymal stem cells (MSCs) are primarily isolated by their adherence to plastic and their in vitro growth characteristics. Expansion of these cells from an adherent culture is the only method to obtain a sufficient number of cells for use in clinical practice and research. However, little is known with regard to the effect of adherence to plastic on the phenotype of the cells. In the present study, bone marrow CD45-CD31-CD44- stem cell antigen (Sca)-1+ MSCs were sorted by flow cytometry and expanded in adherent cultures. The expression levels of the adhesion molecule, Sca-1, in the adherent cultures were compared with those from nonadherent cultures at different time points. The flow cytometry results indicated that the expression levels of Sca-1 decreased in the MSCs in the nonadherent cultures grown in ultra-low-adherent plates. Furthermore, the result was confirmed by quantitative polymerase chain reaction at the same time points. Therefore, the results demonstrated that the loss of plastic adherence downregulated the expression of Sca-1. The observations may provide novel insights into the molecular mechanisms underlying plastic adherent culture.

Certain limitations of the neurosphere assay (NSA) have resulted in a search for alternative culture techniques for brain tumor-initiating cells (TICs). Recently, reports have described growing glioblastoma (GBM) TICs as a monolayer using laminin. We performed a side-by-side analysis of the NSA and laminin (adherent) culture conditions to compare the growth and expansion of GBM TICs. GBM cells were grown using the NSA and adherent culture conditions. Comparisons were made using growth in culture, apoptosis assays, protein expression, limiting dilution clonal frequency assay, genetic affymetrix analysis, and tumorigenicity in vivo. In vitro expansion curves for the NSA and adherent culture conditions were virtually identical (P=0.24) and the clonogenic frequencies (5.2% for NSA vs. 5.0% for laminin, P=0.9) were similar as well. Likewise, markers of differentiation (glial fibrillary acidic protein and beta tubulin III) and proliferation (Ki67 and MCM2) revealed no statistical difference between the sphere and attachment methods. Several different methods were used to determine the numbers of dead or dying cells (trypan blue, DiIC, caspase-3, and annexin V) with none of the assays noting a meaningful variance between the two methods. In addition, genetic expression analysis with microarrays revealed no significant differences between the two groups. Finally, glioma cells derived from both methods of expansion formed large invasive tumors exhibiting GBM features when implanted in immune-compromised animals. A detailed functional, protein and genetic characterization of human GBM cells cultured in serum-free defined conditions demonstrated no statistically meaningful differences when grown using sphere (NSA) or adherent conditions. Hence, both methods are functionally equivalent and remain suitable options for expanding primary high-grade gliomas in tissue culture.