Internet for Pharmaceutical and Biotech Communities
| Newsletter | Advertising |
 
 
 

  

Pharm/Biotech
Resources

Outsourcing Guide

Cont. Education

Software/Reports

Training Courses

Web Seminars

Jobs

Buyer's Guide

Home Page

Pharm Patents /
Licensing

Pharm News

Federal Register

Pharm Stocks

FDA Links

FDA Warning Letters

FDA Doc/cGMP

Pharm/Biotech Events

Consultants

Advertiser Info

Newsletter Subscription

Web Links

Suggestions

Site Map
 

 
   



 

Title:  Method of forming vascularized kidney tissue
United States Patent: 
7,074,552
Issued: 
July 11, 2006

Inventors: 
Nigam; Sanjay Kumar (Del Mar, CA); Qiao; Jizeng (Lexington, MA); Sakurai; Hiroyuki (San Diego, CA)
Assignee: 
The Regents of the University of California (Oakland, CA)
Appl. No.:  595195
Filed: 
June 16, 2000


 

Covidien Pharmaceuticals Outsourcing


Abstract

A method for constructing a stable bioactive mammalian embryonic kidney is described herein. A kidney so constructed requires no artificial support, nor porous man made membranes or tubing to effectuate its biological function of filtering body fluids. A single donor embryonic kidney, or fragment thereof, can produce a great number of functional kidneys suitable for treating subjects with various kidney disorders. It is anticipated that said in vitro produced kidney would be less, or not at all, antigenic when transplanted into a subject, because of its embryonic character and artificial propagation in culture. This method of producing a functional organ can be useful in cloning other organ structures containing inducible epithelial tissues.

SUMMARY OF THE INVENTION

The primary object of this invention is to provide functioning replacement organs or functional fragments thereof that are suitable for transplanting into recipients suffering from a variety of life-threatening diseases or developmental anomalies.

Another object in accordance with the present invention is to generate functional mammalian epithelium-derived organs, or active fragments thereof from embryonic explants, tissues or cells utilizing in vitro culture techniques.

Another object of this invention is to define soluble inducing factors effective in transforming embryonic epithelial cells or tissues into regenerating functional organs, glands and the like.

A further, most preferred object is to provide a bank of embryonic organs and tissues capable of replacing diseased, or otherwise incapacitated vital organs and tissues, minimizing the need for matching donors and/or immunosuppressive drugs.

In accordance with these objects, this invention contemplates a method for constructing a functional mammalian tubulogenic organ or fragment thereof in vitro. The method involves culturing and propagating embryonic explants, tissues or cells by isolating said explants, tissues or cells and growing them in culture with specific soluble and insoluble inducers for sufficient periods of time to allow the cultured specimens to form multiple branches. The tips of these branches are then dissected out and recultured in the presence of serum, growth factor mix, mixture of conditioned and nutrient-rich medium for several generations to form 3-dimensional tubulogenic structures with multiple growing tips. This process can proceed ad infinitum under proper culture conditions having effective inducer substances.

The contemplated method further involves culturing and propagating embryonic mesenchymal tissues capable of inducing limited differentiation and directional growth to form functional organs or tissues. The mesenchymal or other inducing tissue fragments are dissected out at the time of induction, and cultured in the presence of serum, growth factor mix, and a mixture of appropriate conditioned medium and nutrient-rich medium. After several passages in primary culture, growing inductive tissue may be partitioned into multiple fragments. Each fragment can then grown separately in culture. Vasculogenesis within each fragment is induced by substrate deprivation and/or the addition of specific soluble factors.

Finally, a grown, vascularized tissue fragment is combined in coculture with a cultured tubulogenic fragment described hereinabove, in a matrix in which in vitro angiogenesis has begun. The two tissue fragments are grown in nutrient-rich medium conditions to enable continued vasculogenesis. Alternatively, the "cloned" kidney can be implanted for in vivo vascularization.

A more specific and preferred embodiment of this invention is a method for generating a functional mammalian kidney in vitro by culturing and propagating ureteric bud tissue. This method comprises isolating embryonic kidney rudiments by dissection, isolating ureteric bud tissue fragments from mesenchyme by incubating the kidney rudiments with a proteolytic enzyme in the presence of DNAase and/or by mechanical separation. The isolated ureteric bud fragments are suspended in a gel matrix and the gel/fragment composition is placed on porous polycarbonate membrane inserts in wells of tissue culture plates. Growth factors are added to the culture wells, and the gel composition comprising the bud fragments is maintained at the interface of air and medium until the fragments form multiple tubular branches inside the gel matrix. Individual distal branch tips formed during culture are dissected out and recultured in the presence of serum, growth factor mix, mixture of mesenchymal and ureteric bud cell conditioned medium and nutrient-rich medium for several generations.

The mechanical separation of tissue fragments can be accomplished by manual dissection or laser separation and capture. The growth factor mix includes glial cell line-derived neurotrophic factor or functional equivalent thereof. The added conditioned medium contains a heretofore-unidentified growth promoting constituent and/or inducer of differentiation. The extracellular matrix gel comprises a mixture of type I collagen and Matrigel or a comparable support matrix.

An equally preferred embodiment in accordance with this invention is method for simultaneous in vitro culturing and propagation of metanephric mesenchyme. This method comprises dissecting out fetal kidney mesenchyme tissue at the time of induction, culturing fragments of the mesenchymal tissue in the presence of serum, growth factor mix, mixture of mesenchymal and bud cell conditioned medium and nutrient-rich medium, and partitioning the cultured mesenchyme into multiple pieces. Each piece is grown separately in culture for several generations and grown mesenchyme is then subjected to substrate deprivation and/or additional growth factors in order to induce vasculogenesis.

A most preferred embodiment in accordance with this invention is a method for in vitro engineering and constructing a functioning mammalian kidney by culturing and propagating an isolated ureteric bud, permitting the cultured bud to form multiple branches, dissecting out the individual branch tips, and reculturing in the presence of serum, growth factor mix, mixture of mesenchymal and bud cell conditioned medium and nutrient-rich medium for several generations. The method also comprises simultaneously culturing and propagating isolated embryonic or fetal metanephric mesenchyme by dissecting out fetal mesenchyme at the time of induction, culturing mesenchymal tissue in the presence of serum, growth factor mix, mixture of mesenchymal and bud cell conditioned medium and nutrient-rich medium, potentially partitioning the mesenchyme into multiple pieces with the option of growing each piece separately, and inducing vasculogenesis by subjecting grown mesenchyme to substrate deprivation. The most preferred method then provides for recombining each vascularized mesenchyme piece with each cultured bud in a matrix in which in vitro angiogenesis has begun, and growing in richest medium conditions to ensure continued vasculogenesis.

Thus, in the most preferred embodiment, is a functional mammalian kidney constructed from isolated embryonic or fetal kidney tissue or cells cultured in rich medium that has present a mixture of growth factors and inducer substances, and comprises recombination of an isolated ureteric bud propagated in culture to produce a functioning nephron, and metanephric mesenchyme propagated from cultured embryonic mesenchymal tissue fragments or cells. Said mesenchyme has the capability of inducing differentiation and providing directional guidance to the branching tubulogenic bud.
 


Claim 1 of 5 Claims

1. A method for constructing a mammalian tissue or a fragment thereof in vitro, comprising: (a) culturing and propagating embryonic epithelial-derived explants, tissue or cells comprising: (i) isolating the tissues or cells and growing them in culture, (ii) permitting the tissue or cells to form multiple branches, (iii) dissecting out individual branch tips, (iv) culturing the individual branch tips in the presence of nutrient medium, serum, at least one growth factor, and BSN-conditioned medium (BSN-CM) on an extracellular matrix (ECM) gel for several generations to generate branch tip buds; (b) culturing and propagating isolated embryonic or fetal metanephric mesenchyme comprising: (i) dissecting out embryonic or fetal metanephric mesenchyme at the time of induction, (ii) culturing the embryonic or fetal metanephric mesenchymal tissue in the presence of nutrient medium, serum, at least one growth factor, and BSN-CM, (iii) partitioning mesenchyme into multiple pieces and culturing each piece separately, and (iv) inducing vasculogenesis by subjecting cultured mesenchyme to substrate deprivation or addition of soluble growth factors; (c) combining each vascularized mesenchyme with each cultured branch tip bud in a matrix in which in vitro angiogenesis has begun such that the mesenchyme and tip bud are in close contact; and (d) culturing the combined tissue under conditions to ensure continued cell growth to obtain a vascularized mammalian tissue, wherein the at least one growth factor comprises glial cell line-derived neurotrophic factor (GDNF).

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
[ Outsourcing Guide ] [ Cont. Education ] [ Software/Reports ] [ Training Courses ]
[ Web Seminars ] [ Jobs ] [ Consultants ] [ Buyer's Guide ] [ Advertiser Info ]

[ Home ] [ Pharm Patents / Licensing ] [ Pharm News ] [ Federal Register ]
[ Pharm Stocks ] [ FDA Links ] [ FDA Warning Letters ] [ FDA Doc/cGMP ]
[ Pharm/Biotech Events ] [ Newsletter Subscription ] [ Web Links ] [ Suggestions ]
[ Site Map ]