Method for expansion of stem cells

Inactive Publication Date: 2006-08-17
RIORDAN NEIL H
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0083] In an additional embodiment of the present invention, a method of culturing a placenta in its original 3-dimensional structure is provided, in such a manner as to reproduce the in vivo environment in which it resides in the pregnant woman, thus retaining capability of generation and secretion of growth factors and proteins that maintain the fetal regenerative capacity. The method involves acquiring a placenta under sterile conditions, cannulating blood vessels of the placenta in order to allow proper perfusion in circumstances similar to as if the placenta was performing its in vivo functions, perfusing the placenta with a nutrient mix in a buffer that would mimic physiological conditions, maintaining a temperature and physical environment similar to that found in the pregnant woman's body, and imitating conditions of flow, pH, oxygenation, and pressure similar to that found in the body. The perfusion of both the maternal and fetal circulatory components of the placenta can be performed. A nutrient mixture can be used that possesses similar nutrient requirements as the fetal and maternal circulation, respectively. A temperature of 37° C. can be maintained during the perfusion process. The pH can be monitored, for example, by the perfusion apparatus in a real-time basis, and adjusted using adequate quantities of acids, bases, or buffers. The oxygen content can be maintained similar to that found in the fetal and maternal circulatory contribution to the placenta. The oxygen content may

Problems solved by technology

Unfortunately, many such methodologies involve the use of either murine feeder cells or other animal components, hence limiting the therapeutic potential of these cells.
The dose limiting variable in cancer chemotherapy is bone marrow toxicity.
Unfortunately, wide spread use of bone marrow induced tolerance is limited by the fact that bone marrow transplantation is associated with a high degree of morbidity and mortality during the myeloablative phase.
In addition, the possibility of graft versus host disease is another pitfall to the full-scale implementation.
Specifically, it is known that the process of autoimmunity requires the failure of several self-tolerance mechanisms before clinical presentation appears.
During autoimmunity the failure of all of these systems is usually a culmination of environmental and genetic factors occurring over a protracted period of time.
Induction of tolerance through hematopoietic stem cell transplantation, either from bone marrow or peripheral blood sources possesses the intrinsic danger of bone marrow failure during ablation of the recipient immune system.
Although non-myeloablative protocols are under development, even these carry the risk of immune suppression due to the lymphoablation.
Unfortunately, mesenchymal cell expansion is

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Incubation of Placenta in Growth Medium

[0130] A fresh human placenta obtained from vaginal delivery was placed in a sterile plastic container. The placenta was rinsed with an anticoagulant solution comprising phosphate buffered saline (Gibco-Invitrogen, Grand Island, N.Y.), containing a 1:1000 concentration of heparin (1% w / w) (American Pharmaceutical Partners, Schaumburg, Ill.).

[0131] The placenta was then covered with a DMEM media (Gibco) in a sterile container such that the entirety of the placenta was submerged in said media, and incubated at 37° C. in a humidified 5% CO2 incubator for 24 hours. At the end of the 24 hours, the live placenta conditioned medium (LPCM) was isolated from the container and sterile-filtered using a commercially available sterile 0.2 micron filter (VWR).

example 2

Isolation of CD 34+ cells from Human Umbilical Cord Blood and Subsequent Growth of Cells

[0132] Approximately 40 ml of cord blood was collected from a human umbilical cord via venipuncture and allowed to drop by gravitational force into a 250 ml sterile bag containing 20 ml citrate-phosphate-dextrose under sterile conditions. Collected blood cells were layered onto 50 ml conical tubes containing Ficoll-Hypaque (density 1.077 gram / ml; Sigma, St Louis, Mo.) and centrifuged at 400×g for 30 minutes. The mononuclear cells in the interface layer were then collected, washed three times in PBS, and re-suspended in PBS solution containing 0.5% serum albumin. CD34+ cells were purified from the mononuclear cell fraction by immuno-magnetic separation using the Magnetic Activated Cell Sorting (MACS) CD34+ Progenitor Cell Isolation Kit (Miltenyi-Biotec, Auburn, Calif.) according to manufacturer's recommendations. The purity of the CD34+ cells obtained ranged between 95% and 98%, based on Flow Cyt...

example 3

Expansion of Stem Cells

[0133] At the end of the 24 hour period, the LPCM from Example 1 was added to the wells of the sterile 24 well tissue culture plate in a volume of 0.25 ml. Umbilical cord mononuclear cells harvested as described in Example 1 were resuspended in DMEM in a volume of 0.25 ml and added to the wells containing LPCM. The final concentration of mononuclear cells was 10×106 cells per ml. The cultures were subsequently incubated for an additional seven days at 37° C. in a humidified 5% CO2 incubator. The number of CD 34+ cells and viability was then determined by flow cytometry as described in Example 2 both at the beginning of cell culture and subsequently after 7 days of culture. The number of viable CD34+ cells had increased 27.4 fold over the starting number of cells. In contrast, cells that were cultured with DMEM media alone in absence of LPCM had a decline in viable CD34+ cell numbers by approximately 7 fold.

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Abstract

A method of increasing the growth of stem cells by mixing the stem cells with a growth medium that has been conditioned by an incubation with placental tissue. The method increases the expansion of the stem cell population.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority under 35 U.S.C. § 119 to U.S. Provisional Application No. 60 / 653,390, which was filed on Feb. 15, 2005, the disclosure of which is hereby incorporated by reference in its entirety.FIELD OF THE INVENTION [0002] This invention relates to the field of stem cell technology. More particularly, the invention describes a new method for increasing the growth of stem cells by mixing the stem cell culture with a medium that has been incubated with placental tissue. BACKGROUND OF THE INVENTION [0003] Stem cells have the ability to divide for indefinite periods in culture and to give rise to specialized cells. Typically, stem cells are divided into two main groups: adult stem cells and embryonic stem cells. Stem cells may also be generated through artificial means such as nuclear transfer, cytoplasmic transfer, cell fusion, parthenogenesis and reprogramming. Isolated stem cells can give rise to many types of differe...

Claims

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Application Information

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IPC IPC(8): A61K35/14C12N5/08A61K35/28A61K35/44A61K35/50A61K35/51
CPCA61K8/982A61K35/50A61K35/51A61K35/44A61K35/28A61K38/1825C12N2502/02C12N5/0018A61Q19/08A61Q7/00A61K2300/00
Inventor RIORDAN, NEIL H.
Owner RIORDAN NEIL H
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