culture-methods-to-study-apical-specific-interactions-using

Culture Methods to Study Apical-Specific Interactions using Intestinal Organoid Models

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to ...

Murine embryonic day 14 or E14 neural stem cells (NSCs), first isolated and characterized as a stem cell in culture, are a unique population of cells capable of self-renewal. In addition, they produce a large number of progeny capable of differentiating into the three primary phenotypes-neurons, astrocytes, and oligodendrocytes-found in the adult mammalian central nervous system (CNS). A defined serum-free medium supplemented with epidermal growth factor (EGF) is used to maintain the NSCs in an undifferentiated state in the form of clusters of cells, called neurospheres, for several culture passages. When EGF is removed and serum added to the medium, the intact or dissociated neurospheres differentiate into the three primary CNS phenotypes. This chapter outlines the simple NSC culture methodology and provides some of the more important details of the assay to achieve reproducible cultures.

Generation and differentiation of neurospheres from murine embryonic day 14 central nervous system tissue.

Generierung und Differenzierung von Neurosph&#xE4;ren aus murinen embryonalen Tag 14 Gewebe des zentralen Nervensystems.

Advancement in our understanding of the biology of adult stem cells and their therapeutic potential relies heavily on meaningful functional assays that can identify and measure stem cell activity in vivo and in vitro. In the mammalian nervous system, neural stem cells (NSCs) are often studied using a culture system referred to as the neurosphere assay. We previously challenged a central tenet of this assay, that all neurospheres are derived from a NSC, and provided evidence that it overestimates NSC frequency, rendering it inappropriate for quantitation of NSC frequency in relation to NSC regulation. Here we report the development and validation of the neural colony-forming cell assay (NCFCA), which discriminates stem from progenitor cells on the basis of their proliferative potential. We anticipate that the NCFCA will provide additional clarity in discerning the regulation of NSCs, thereby facilitating further advances in the promising application of NSCs for therapeutic use.

Enumeration of neural stem and progenitor cells in the neural colony-forming cell assay.

Fortschritt im Verst&auml;ndnis der Biologie der adulten Stammzellen und ihr therapeutisches Potential beruht stark auf sinnvolle funktionale Assays, die identifizieren und Stammzell-Aktivit&auml;t in vivo und in vitro zu messen k&ouml;nnen. S&auml;ugetier-Nervensystems studierte neuraler Stammzellen (NSCs) sind oft mit einem Kultursystem als Neurosphere Assays bezeichnet. Wir herausgefordert zuvor eine zentrale Lehre dieser Probe, dass alle Neurospheres von einem NSC abgeleitet, und legte Beweise daf&uuml;r vor, dass es &uuml;bersch&auml;tzt NSC-Frequenz, was es f&uuml;r die quantitative Bestimmung der NSC-Frequenz bei NSC Verordnung ungeeignet. Hier berichten wir die Entwicklung und Validierung von der neuronalen kolonienbildende Zellenprobe (NCFCA), die Stammzellen aus Vorl&auml;uferzellen auf der Grundlage ihrer proliferative Potential benachteiligt werden. Wir erwarten, dass die NCFCA bieten zus&auml;tzliche Klarheit in die Regulierung der NSCs anspruchsvolle, und erleichtert so weiter Fortschritte in die vielversprechende Anwendung der NSCs f&uuml;r therapeutische Zwecke.

Enumeration von Stamm- und Vorl&auml;uferzellen von Nervenzellen in der neuronalen kolonienbildende Zellenprobe.

Recent reports have highlighted several parameters of the neurosphere culture or assay system which render it unreliable as a quantitative in vitro assay for measuring neural stem cell (NSC) frequency. The single-step semi-solid based assay, the Neural Colony Forming Cell (NCFC) assay is an assay which was developed to overcome some of the limitations of the neurospheres assay in terms of accurately measuring NSC numbers. The NCFC assay allows the discrimination between NSCs and progenitors by the size of colonies they produce (i.e. their proliferative potential). The NCFC assay and other improved tissue culture tools offer further advances in the promising application of NSCs for therapeutic use. 

Enumerating stem cell frequency: neural colony forming cell assay.

Although regenerative medicine products are at the forefront of scientific research, technological innovation, and clinical translation, their reproducibility and large-scale production are compromised by automation, monitoring, and standardization issues. To overcome these limitations, new technologies at software (e.g., algorithms and artificial intelligence models, combined with imaging software and machine learning techniques) and hardware (e.g., automated liquid handling, automated cell expansion bioreactor systems, automated colony-forming unit counting and characterization units, and scalable cell culture plates) level are under intense investigation. Automation, monitoring and standardization should be considered at the early stages of the developmental cycle of cell products to deliver more robust and effective therapies and treatment plans to the bedside, reducing healthcare expenditure and improving services and patient care.

Automation, Monitoring, and Standardization of Cell Product Manufacturing.

Research on chronic and acute myeloid leukemia (CML/AML) is focused on the development of novel therapeutic strategies to eliminate leukemic stem/progenitor cells that are responsible for drug resistance and disease relapse. Methods to culture hematopoietic stem/progenitor cells (HSPCs) from blood or bone marrow samples are indispensable for investigating disease pathogenesis and delineating drug responses in individual patients. A key challenge in this area is that primary leukemic cells grow poorly in culture or rapidly differentiate and lose their hematopoietic potential. Access to patient samples can also be limiting or cell numbers too low to enable large-scale assays and/or to obtain reproducible quantitative data. Here we describe a feeder cell-free and serum-free liquid culture system for the expansion of CD34 HSPCs from CML/AML samples and healthy control tissues. Following 7 or 14 days of culture, CD34 cells are expanded 30- to 65-fold or 400- to 800-fold, yielding a purity of ∼80% and ∼60% CD34 cells, respectively. This system was adapted to a 96-well format to measure the sensitivity of leukemic and normal HSPCs to cytotoxic drugs after only 7 days. The assay requires only 10 cells per well to determine drug IC values and can be performed with uncultured and culture-expanded cells. Importantly, resulting IC values strongly correlate with those obtained in the classic colony-forming unit (CFU) assay. Compared with the CFU assay, this novel 96-well liquid-based assay designed specifically for leukemic and normal HSPCs is faster and simpler, with more flexible readout methods for selecting candidates for further drug development.

Article	Year
Generation and differentiation of neurospheres from murine embryonic day 14 central nervous system tissue. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 15361668	2005
Enumeration of neural stem and progenitor cells in the neural colony-forming cell assay. Stem cells (Dayton, Ohio)\| PubMed ID: 18218818	2008
Enumerating stem cell frequency: neural colony forming cell assay. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 23934839	2013
Automation, Monitoring, and Standardization of Cell Product Manufacturing. Frontiers in bioengineering and biotechnology\| PubMed ID: 32766229	2020
Feeder-free and serum-free in vitro assay for measuring the effect of drugs on acute and chronic myeloid leukemia stem/progenitor cells. Experimental hematology\| PubMed ID: 32798646	2020

Sharon Louis

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