Isoleucine transport by cultured human fibroblasts. II. Selection of a cell line with reduced isoleucine uptake

Amphotropic pseudotyped retroviral vectors have typically been used to infect target cells without prior concentration. Although this can yield high rates of infection, higher rates may be needed where highly efficient coinfection of two or more vectors is needed. In this investigation we used amphotropic retroviral vectors produced by the Plat-A cell line and studied coinfection rates using green and red fluorescent proteins (EGFP and dsRed2). Target cells were primary human fibroblasts (PHF) and 3T3 cells. Unconcentrated vector preparations produced a coinfection rate of∼4% (defined as cells that are both red and green as a percentage of all cells infected). Optimized spinoculation, comprising centrifugation at 1200 g for 2 hours at15∘C, increased the coinfection rate to∼10%. Concentration by centrifugation at 10,000 g or by flocculation using Polybrene increased the coinfection rate to∼25%. Combining the two processes, concentration by Polybrene flocculation and optimized spinoculation, increased the coinfection rate to 35% (3T3) or>50% (PHF). Improved coinfection should be valuable in protocols that require high transduction by combinations of two or more retroviral vectors.

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Biosynthesis of electron transfer flavoprotein in a cell-free system and in cultured human fibroblasts. Defect in the alpha subunit synthesis is a primary lesion in glutaric aciduria type II.

Journal of Clinical Investigation ◽

10.1172/jci112691 ◽

1986 ◽

Vol 78 (4) ◽

pp. 997-1002 ◽

Cited By ~ 38

Author(s):

Y Ikeda ◽

S M Keese ◽

K Tanaka

Keyword(s):

Human Fibroblasts ◽

Glutaric Aciduria Type ◽

Primary Lesion ◽

Alpha Subunit ◽

Type Ii ◽

Free System ◽

Glutaric Aciduria ◽

Cultured Human Fibroblasts ◽

A Cell ◽

Glutaric Aciduria Type Ii

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Aberrant Type C Virus Production in a Cell Line Derived from Balb/3T3

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100094693 ◽

1972 ◽

Vol 30 ◽

pp. 286-287

Author(s):

N. Savage ◽

A. Hackett

Keyword(s):

Electron Microscopy ◽

Cell Line ◽

Leukemia Virus ◽

Morphological Characteristics ◽

Virus Production ◽

Oncogenic Viruses ◽

Endogenous Virus ◽

Virus Particles ◽

Moloney Leukemia Virus ◽

A Cell

A cell line, UC1-B, which was derived from Balb/3T3 cells, maintains the same morphological characteristics of the non-transformed parental culture, and shows no evidence of spontaneous virus production. Survey by electron microscopy shows that the cell line consists of spindle-shaped cells with no unusual features and no endogenous virus particles.UC1-B cells respond to Moloney leukemia virus (MLV) infection by a change in morphology and growth pattern which is typical of cells transformed by sarcoma virus. Electron microscopy shows that the cells are now variable in shape (rounded, rhomboid, and spindle), and each cell type has some microvilli. Virtually all (90%) of the cells show virus particles developing at the cell surface and within the cytoplasm. Maturing viruses, typical of the oncogenic viruses, are found along with atypical tubular forms in the same cell.

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Combination of ribosome and phage display for fast selection of high affinity VHH antibody fragments

Bulletin of Russian State Medical University ◽

10.24075/brsmu.2019.002 ◽

2019 ◽

pp. 27-33

Author(s):

YuE Kravchenko ◽

SV Ivanov ◽

DS Kravchenko ◽

EI Frolova ◽

SP Chumakov

Keyword(s):

Phage Display ◽

Selection Procedure ◽

Free System ◽

Phage Displays ◽

High Affinity ◽

Binding Domains ◽

A Cell ◽

Vhh Antibodies ◽

Efficiency Of Selection ◽

Selection Of

Selection of antibodies using phage display involves the preliminary cloning of the repertoire of sequences encoding antigen-binding domains into phagemid, which is considered the bottleneck of the method, limiting the resulting diversity of libraries and leading to the loss of poorly represented variants before the start of the selection procedure. Selection in cell-free conditions using a ribosomal display is devoid from this drawback, however is highly sensitive to PCR artifacts and the RNase contamination. The aim of the study was to test the efficiency of a combination of both methods, including pre-selection in a cell-free system to enrich the source library, followed by cloning and final selection using phage display. This approach may eliminate the shortcomings of each method and increase the efficiency of selection. For selection, alpaca VHH antibody sequences suitable for building an immune library were used due to the lack of VL domains. Analysis of immune libraries from the genes of the VH3, VHH3 and VH4 families showed that the VHH antibodies share in the VH3 and VH4 gene groups is insignificant, and selection from the combined library is less effective than from the VHH3 family of sequences. We found that the combination of ribosomal and phage displays leads to a higher enrichment of high-affinity fragments and avoids the loss of the original diversity during cloning. The combined method allowed us to obtain a greater number of different high-affinity sequences, and all the tested VHH fragments were able to specifically recognize the target, including the total protein extracts of cell cultures.

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In vitro Three Dimensional Scaffold-free Construct of Human Adipose-derived Stem Cells in Coculture with Endothelial Cells and Fibroblasts

Revista de Chimie ◽

10.37358/rc.17.6.5670 ◽

2017 ◽

Vol 68 (6) ◽

pp. 1341-1344

Author(s):

Grigore Berea ◽

Gheorghe Gh. Balan ◽

Vasile Sandru ◽

Paul Dan Sirbu

Keyword(s):

Tissue Engineering ◽

Stem Cells ◽

Endothelial Cells ◽

Single Cell ◽

Cell Line ◽

Bone Repair ◽

Umbilical Vein ◽

Human Fibroblasts ◽

Three Dimensional ◽

Adipose Derived Stem Cells

Complex interactions between stem cells, vascular cells and fibroblasts represent the substrate of building microenvironment-embedded 3D structures that can be grafted or added to bone substitute scaffolds in tissue engineering or clinical bone repair. Human Adipose-derived Stem Cells (hASCs), human umbilical vein endothelial cells (HUVECs) and normal dermal human fibroblasts (NDHF) can be mixed together in three dimensional scaffold free constructs and their behaviour will emphasize their potential use as seeding points in bone tissue engineering. Various combinations of the aforementioned cell lines were compared to single cell line culture in terms of size, viability and cell proliferation. At 5 weeks, viability dropped for single cell line spheroids while addition of NDHF to hASC maintained the viability at the same level at 5 weeks Fibroblasts addition to the 3D construct of stem cells and endothelial cells improves viability and reduces proliferation as a marker of cell differentiation toward osteogenic line.

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