Selection of kidney cell types in primary glomerular explant outgrowths by in vitro culture conditions

1986 ◽  
Vol 84 (1) ◽  
pp. 69-92
Author(s):  
T.D. Oberley ◽  
A.H. Yang ◽  
J. Gould-Kostka
Keyword(s):  
Epithelial Cell ◽  
Cell Types ◽  
Defined Medium ◽  
Culture Conditions ◽  
Chemically Defined Medium ◽  
Cell Type ◽  
Epithelial Cell Type ◽  
Tubular Cells ◽  
Selection Of

Adult guinea pig glomeruli were grown in vitro either in serum or in a chemically defined medium. Glomeruli were plated either directly into plastic flasks or into plastic flasks that had been coated with the extracellular matrix produced by the PF-HR-9 mouse teratocarcinoma endodermal cell line. Both the composition of the medium and the nature of the culture substrate affected whole glomerular attachment and the type of cells produced in culture. Quantitative studies demonstrated selection of cell types by different culture conditions. Three colony types, each composed of distinctive cell types, could be identified by morphological features. The cells constituting two of these colony types were epithelial in nature, but they were identified as different epithelial types by both histochemical and ultrastructural criteria. Previous studies suggested that one epithelial cell type was derived from the glomerular visceral epithelial cell. This study demonstrates that this cell type could be selectively grown in defined medium on plastic. A second cell type showed several features of renal tubular epithelial cells, including histochemical staining for catalase, cell surface microvilli and cilia, and formation of hemicysts and structures that resembled tubules after prolonged periods in culture. To demonstrate that the ‘glomerulus-derived’ tubular cells were indeed tubular epithelium, we isolated purified renal cortical tubules (greater than 99% pure) and cultured them on the HR-9 matrix in a serum-free chemically defined medium. The resultant outgrowths had morphological properties identical to those of the glomerulus-derived tubular cells. It seems likely that small tubular fragments attached to a minority of the glomeruli are the source of these glomerulus-derived tubular cells. Neither epithelial cell type could be subcultured on plastic, but both could be passaged on the HR-9 matrix. A third cell type, the spindle-shaped cell, was easily propagated on both plastic and the HR-9 matrix. The origin of this cell type is not clear. Our results demonstrate the important effect of culture conditions on the selection, growth and differentiation of kidney cell types in vitro.

A novel epithelial cell from neonatal rat lung: isolation and differentiated phenotype

1990 ◽  
Vol 259 (6) ◽  
pp. L415-L425 ◽  
Author(s):  
P. E. Roberts ◽  
D. M. Phillips ◽  
J. P. Mather
Keyword(s):  
Epithelial Cell ◽  
Molecular Mechanisms ◽  
Basal Medium ◽  
Fold Increase ◽  
Cell Types ◽  
Cell Number ◽  
Neonatal Rat ◽  
Rat Lung ◽  
Cell Type

A novel epithelial cell from normal neonatal rat lung has been isolated, established, and maintained for multiple passages in the absence of serum, without undergoing crisis or senescence. By careful manipulation of the nutrition/hormonal microenvironment, we have been able to select, from a heterogeneous population, a single epithelial cell type that can maintain highly differentiated features in vitro. This cell type has characteristics of bronchiolar epithelial cells. A clonal line, RL-65, has been selected and observed for greater than 2 yr in continuous culture. It has been characterized by ultrastructural, morphological, and biochemical criteria. The basal medium for this cell line is Ham's F12/Dulbecco's modified Eagle's (DME) medium plus insulin (1 micrograms/ml), human transferrin (10 micrograms/ml), ethanolamine (10(-4) M), phosphoethanolamine (10(-4) M), selenium (2.5 x 10(-8) M), hydrocortisone (2.5 x 10(-7) M), and forskolin (5 microM). The addition of 150 micrograms/ml of bovine pituitary extract to the defined basal medium stimulates a greater than 10-fold increase in cell number and a 50- to 100-fold increase in thymidine incorporation. The addition of retinoic acid results in further enhancement of cell growth and complete inhibition of keratinization. We have demonstrated a strategy that may be applicable to isolating other cell types from the lung and maintaining their differentiated characteristics for long-term culture in vitro. Such a culture system promises to be a useful model in which to study cellular events associated with differentiation and proliferation in the lung and to better understand the molecular mechanisms involved in these events.

scholarly journals Paediatric gastric organoids as a tool for disease modelling and clinical translation

2021 ◽  
Vol 37 (3) ◽  
pp. 317-324
Author(s):  
Brendan C. Jones ◽  
Giuseppe Calà ◽  
Paolo De Coppi ◽  
Giovanni Giuseppe Giobbe
Keyword(s):  
Epithelial Cell ◽  
Single Cell ◽  
3D Culture ◽  
Cell Types ◽  
Defined Medium ◽  
Gastric Epithelium ◽  
Apical Surface ◽  
Gastric Glands ◽  
Cell Dissociation

Abstract Purpose Knowledge of gastric epithelial homeostasis remains incomplete, lacking human-specific models for study. This study establishes a protocol for deriving gastric epithelial organoids from paediatric gastric biopsies, providing a platform for modelling disease and developing translational therapies. Methods Full-thickness surgical samples and endoscopic mucosal biopsies were obtained from six patients. Gastric glands were isolated by a chemical chelation protocol and then plated in 3D culture in Matrigel® droplets in chemically defined medium. After formation, organoids were passaged by single cell dissociation or manual disaggregation. Cell composition and epithelial polarity of organoids were assessed by bright field microscopy and immunofluorescence analysis, comparing them to native paediatric gastric tissue. Results Gastric glands were successfully isolated from all six patients who were aged 4 months to 16 years. Gastric glands from all patients sealed to form spherical gastric organoids. These organoids could be passaged by manual disaggregation or single cell dissociation, remaining proliferative up to 1 year in culture. Organoids retained normal epithelial cell polarity, with the apical surface orientated towards the central lumen. Organoids expressed markers of mature gastric epithelial cell types, except for parietal cells. Conclusion Gastric organoids can be reliably generated from paediatric biopsies and are a representative in vitro model for studying gastric epithelium.

69 EFFECTS OF CELL TYPE, PRE-ACTIVATION PROTOCOL, AND CULTURE CONDITIONS ON DEVELOPMENT OF PORCINE HANDMADE CLONED EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 193
Author(s):  
J. C. Mezzalira ◽  
L. U. Ohlweiler ◽  
A. Massie ◽  
E. Monaco ◽  
E. P. Silva ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Cytochalasin B ◽  
Cell Types ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Dependent Manner ◽  
Cell Type ◽  
Embryo Production ◽  
Distinct Cell

Despite the rather successful and widespread use of cloning in various species, distinct cell types from the same species and even the same genotype display differences in blastocyst yield. Moreover, variations in the protocol for embryo production can influence development to the blastocyst stage and subsequent fetal development. The aim of this study was to evaluate the effect of 2 cell types and 2 embryo pre-activation protocols with or without the presence of FCS in the in vitro culture medium on development of handmade pig cloned embryos to the blastocyst stage. Cumulus-oocyte complexes recovered from sow ovaries were in vitro-matured for 38 to 40 h. Denuded matured oocytes selected by the presence of a polar body had the zona pellucida removed in a 0.2% protease HEPES-buffered solution +25% FCS, followed by manual bisection and UV screening of enucleated halves using Hoechst stain. Clone embryo reconstruction was performed using a phytohemoagglutinin solution to adhere 2 cytoplasts and a somatic cell. Adipocyte-derived mesenchymal stem cells (ADMSC) from a Yorkshire pig or granulosa cells (GC) from an Ossabaw pig were used as nuclear donors. Following electrical fusion, couplets were pretreated with a brief exposure to cytochalasin B (CB) or cytochalasin B + cycloheximide (CB+CX) in the presence of serum before the electrical activation (Naruse et al. 2007 Theriogenology 68, 709-716; Du et al. 2009 Reprod. Fertil. Dev. 21, 114). Activated embryos were in vitro-cultured in the well of the well (WOW) system, with 2 embryos per microwell, for 7 days in PZM-3 medium +0.3% BSA in the presence (FBS+) or absence (FBS-) of 10% FCS. Cleavage (Day 2, chi-square test) and blastocyst (Day 7, Fisher test) rates, on a per WOW basis, were compared for a level of significance of 5%. Our preliminary data indicate that the presence of serum in the IVC affected cleavage and blastocyst yield in a cell-type-dependent manner. The presence of serum enhanced the blastocyst yield for ADMSC, whereas for GC, only the absence of serum allowed any blastocyst development. The cell type and the pre-activation protocol did not appear to affect cleavage and embryo development to the blastocyst stage. Despite the low number of replications, our results reinforce the importance of optimizing the embryo production system taking into consideration the individual requirements for distinct cell types, procedures, and culture conditions. Table 1.Effects of cell type, pre-activation process and in vitro culture (IVC) medium on development of handmade pig cloned embryos

scholarly journals Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1–9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical Neurons

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Thu T. Duong ◽  
James Lim ◽  
Vidyullatha Vasireddy ◽  
Tyler Papp ◽  
Hung Nguyen ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Transgene Expression ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Cell Types ◽  
Egfp Expression ◽  
Cell Type ◽  
Egfp Gene ◽  
Rat Cortical Neurons

Recombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ acrossin vitroand ex vivo cellular transduction strategies. This study compared 11 rAAV serotypes, carrying one reporter transgene cassette containing a cytomegalovirus immediate-early enhancer (eCMV) and chicken beta actin (CBA) promoter driving the expression of an enhanced green-fluorescent protein (eGFP) gene, which was transduced into four different cell types: human iPSC, iPSC-derived RPE, iPSC-derived cortical, and dissociated embryonic day 18 rat cortical neurons. Each cell type was exposed to three multiplicity of infections (MOI: 1E4, 1E5, and 1E6 vg/cell). After 24, 48, 72, and 96 h posttransduction, GFP-expressing cells were examined and compared across dosage, time, and cell type. Retinal pigmented epithelium showed highest AAV-eGFP expression and iPSC cortical the lowest. At an MOI of 1E6 vg/cell, all serotypes show measurable levels of AAV-eGFP expression; moreover, AAV7m8 and AAV6 perform best across MOI and cell type. We conclude that serotype tropism is not only capsid dependent but also cell type plays a significant role in transgene expression dynamics.

scholarly journals In vitro mouse spermatogenesis with an organ culture method in chemically defined medium

PLoS ONE ◽  
2018 ◽  
Vol 13 (2) ◽  
pp. e0192884 ◽  
Author(s):  
Hiroyuki Sanjo ◽  
Mitsuru Komeya ◽  
Takuya Sato ◽  
Takeru Abe ◽  
Kumiko Katagiri ◽  
...  
Keyword(s):  
Organ Culture ◽  
Culture Method ◽  
Defined Medium ◽  
Chemically Defined Medium

On the Presence of a Ciliated Columnar Epithelial Cell Type within the Bovine Cervical Mucosa

1973 ◽  
Vol 36 (5) ◽  
pp. 936-940 ◽  
Author(s):  
R. J. Wordinger ◽  
J. B. Ramsey ◽  
J. F. Dickey ◽  
J. R. Hill
Keyword(s):  
Epithelial Cell ◽  
Cell Type ◽  
Epithelial Cell Type ◽  
Columnar Epithelial Cell ◽  
Cervical Mucosa

scholarly journals CellMap: Characterizing the types and composition of iPSC-derived cells from RNA-seq data

2021 ◽  
Author(s):  
Zhengyu Ouyang ◽  
Nathanael Bourgeois ◽  
Eugenia Lyashenko ◽  
Paige Cundiff ◽  
Patrick F Cullen ◽  
...  
Keyword(s):  
Single Cell ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Model Systems ◽  
Rna Seq ◽  
Cell Type ◽  
Fine Grained ◽  
Single Nucleus ◽  
Induced Pluripotent

Induced pluripotent stem cell (iPSC) derived cell types are increasingly employed as in vitro model systems for drug discovery. For these studies to be meaningful, it is important to understand the reproducibility of the iPSC-derived cultures and their similarity to equivalent endogenous cell types. Single-cell and single-nucleus RNA sequencing (RNA-seq) are useful to gain such understanding, but they are expensive and time consuming, while bulk RNA-seq data can be generated quicker and at lower cost. In silico cell type decomposition is an efficient, inexpensive, and convenient alternative that can leverage bulk RNA-seq to derive more fine-grained information about these cultures. We developed CellMap, a computational tool that derives cell type profiles from publicly available single-cell and single-nucleus datasets to infer cell types in bulk RNA-seq data from iPSC-derived cell lines.

Sign in / Sign up

Export Citation Format

Share Document