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Choroid plexus preparation and uses thereof

By using choroid plexus cells or their conditioned media to provide supporting factors to solve the lack of serum use in cell culture media, long-term growth and functional support of multiple cell types is achieved, significantly improving cell survival and proliferation capabilities, and solving The problem of serum-induced death.

Inactive Publication Date: 2008-05-28
NEUROTROPHINCELL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Unfortunately, such purified proteins, such as human serum albumin, suffer from many of the disadvantages of serum
For example, human serum albumin is expensive and regularly deficient, and different sources may differ in the concentration at which the compound is present, thus, leading to unpredictable culture growth and productivity

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Use of CP formulations to support growth and survival of non-neuronal cells in culture

[0088] Free and encapsulated porcine islets were co-cultured with CP cells or CP-conditioned medium, and islet cell viability, maturation, and function were determined under short-term and long-term culture conditions.

[0089] Materials and methods

[0090] Islet cells and isolation and purification

[0091] Porcine islet cells were isolated by digestion of minced pancreas with Liberase via the procedure described by Ricordi et al. (1990), with some modifications. Using aseptic technique, glands were dilated with Liberase (1.5 mg / ml), excess fat, blood vessels and connective tissue were trimmed, minced and digested at 37°C for 10 minutes in a water bath shaking at 120 rpm. After digestion, pass the cells through a sterile 280 μm mesh sieve into a sterile beaker. A second digestion (10 min) was performed on any undigested tissue.

[0092] Further details of the collagenase diges...

Embodiment 2

[0134] CP preparations protect neuronal cells in culture from serum withdrawal-induced cell death

[0135] Choroid plexus (CP) cells secrete a cocktail of neurotrophic factors. In the studies described below, CP-conditioned medium promoted the survival and function of fetal rat neuronal cells in culture, providing protection against neurotrophic deficiency in vitro.

[0136] Materials and methods

[0137] in vitro biological activity

[0138] The in vitro bioactivity of the choroid plexus was determined by placing CP-conditioned medium on primary 15-day embryonic cortical neurons and determining its effect on neuronal survival in the absence of serum. Techniques for preparing and maintaining primary cortical neuronal cultures were similar to those previously described (Fukuda A, Deshpande SB, Shimano Y, Nishino H. "Astrocytes are more vulnerable than neurons to cellular Ca2+ overload induced by a mitochondrial toxin, 3-nitropropionic acid", Neuroscience. 87: 497-507, 1998)....

Embodiment 3

[0148] CP preparations comprising pluripotent neuronal cell precursors

[0149] Choroid plexus (CP) cell clusters comprising cells characteristic of pluripotent neuronal precursors were prepared according to the methods described herein.

[0150] Materials and methods

[0151] cell culture

[0152] The choroid plexuses of neonatal pigs and adult rats were isolated as described herein and cultured in air containing 5% CO 2 and in cell culture in RPMI supplemented with 2% neonatal porcine serum, nicotinamide and cyproxin, as described in Example 1.

[0153] immunochemistry

[0154] Pellet cell clusters (about 2000-2500), remove the medium and reduce it to a volume of 100 microliters, at 35-37 ° C, in Hanks balanced salt buffer solution (200 microliters) suspension with 2% low melting point Agarose mix. The mass was allowed to cool and the solidified agarose block containing the cell clusters was fixed in neutral buffered formalin. The blocks were processed into paraffin us...

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Abstract

The present invention is directed to the use of choroid plexus cells and / or choroid plexus conditioned media for enhancing the growth, survival and / or maintenance of function of non-choroid plexus cells grown in long term or short term culture.

Description

technical field [0001] The present invention relates to the field of media for cell culture and methods for culturing cells in vitro or ex vivo. The invention is applied in the fields of cell culture and tissue transplantation. Background technique [0002] In order to successfully grow cells in vitro or ex vivo, the medium for cell culture must include all the nutrients required to support cell growth and proliferation, including sugars, amino acids, vitamins, salts, and in some cases trace metal ions , purine and pyrimidine. These media are also usually supplemented with animal serum. [0003] Serum is usually derived from fetal bovine, calf or horse and is added to the medium for cell culture at a concentration of 0.5 to 20% v / v. In addition to providing growth-promoting components, serum acts as a carrier / buffer / chelator for unstable or water-insoluble molecules as well as toxin neutralizers, protease inhibitors, cell adhesion promoters, and as an agitated Protective...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/06C12N5/08C12N5/07C12N5/071
CPCC12N2502/08C12N5/0018
Owner NEUROTROPHINCELL
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