Method for growing tissue specimens in vitro

• US 4,940,853 A
• Filed: 07/22/1988
• Issued: 07/10/1990
• Est. Priority Date: 07/22/1988
• Status: Expired due to Term

• Current Assignee: Miriam Hospital (Lifespan Corporation)
• Original Assignee: Herman H. Vandenburgh
• Inventors: Vandenburgh, Herman H.
• Primary Examiner(s): Richman, Barry S.
• Assistant Examiner(s): Beisner, William H.
• Application Number:
  US07/223,292
• Time in Patent Office: 718 Days
• Field of Search: 435/1, 435/240.23, 435/240.241, 435/240.243, 435/284-286
• US Class Current: 435/395
• CPC Class Codes: C12M 25/04 in combination with well or multiwell plates, i.e. culture inserts
• CPC Class Codes: C12M 35/04 Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
• CPC Class Codes: C12N 5/0062 General methods for three-dimensional culture

Abstract

An apparatus and method for growing tissue specimens in vitro wherein the apparatus includes an expandable membrane for receiving a tissue specimen thereon, a mechanism for expanding the membrane and the tissue specimen, and a controller for controlling the expanding mechanism. The controller is operative in accordance with the method for applying an activity pattern to the membrane and a tissue specimen thereon which includes simultaneous continuous stretch activity and repetitive stretch and release activity. The continuous stretch activity and the repetitive stretch and release activity simulate the types of activity to which cells are exposed in vivo due to growth and movement, respectively, and they cause the cells of tissue specimens grown in the apparatus in accordance with the method to develop as three-dimensional structures similar to those grown in vivo.

Claims

1. A method of growing a tissue specimen in vitro, wherein said tissue specimen contains cells which are substantially aligned or require no alignment to simulate in vivo tissue, and said specimen is supported by an expandable support member so that it is expandable therewith comprising the steps of:

(a) maintaining said tissue specimen in a life support environment over the course of said method;

(b) providing sufficient nutrients to sustain said tissue specimen over the course of said method;

(c) maintaining the cells of said tissue specimen in an extracellular matrix sufficient to permit stretching said tissue specimen without causing damage thereto over the course of said method; and

(d) gradually and substantially continuously stretching said support member to stretch said tissue specimen by at least approximately 0.5% over the course of a 24 hr period while periodically repetitively stretching and releasing said support member to stretch and release said tissue specimen by at least approximately 0.01% for a total of at least 60 seconds during the course of said 24 hr period.

2. In the method of claim 1, said tissue specimen being stretched and released by between 0.01% and 60% during said stretch and release activity.

3. In the method of claim 1, said tissue specimen being gradually and substantially continuously stretched at a rate of between 0.005 mm/hr and 1.0 mm/hr.

4. In the method of claim 1, said support member comprising a resilient membrane having a pair of spaced upwardly extending support walls thereon, said support walls being made of a biocompatible material and having opposed support surfaces thereon which have sufficiently roughened textures to permit attachment of said tissue specimen thereto, said support member and tissue specimen being stretched by moving said support walls apart.

5. In the method of claim 1, said tissue specimen being repetitively stretched and released during activity periods of at least five seconds in duration.

6. In the method of claim 5, said activity periods being separated by rest periods of at least five seconds in duration during which said tissue specimen is subjected to said gradual, substantially continuous stretch activity but no significant stretch and release activity.

7. A method of growing a tissue specimen wherein the tissue specimen contains cells requiring alignment to simulate in vivo tissue comprising:

(a) supporting said tissue specimen with an expandable support member, said support member being adapted to permit attachment of said tissue specimen thereto;

(b) maintaining said tissue specimen in a life support environment over the course of said method;

(c) providing sufficient nutrients to sustain said tissue specimen over the course of said method;

(d) maintaining said tissue specimen in an extra-cellular matrix sufficient to permit stretching of said tissue specimen without causing damage thereto over the course of said method;

(e) effecting attachment of said tissue specimen to said support member;

(f) gradually and substantially continuously stretching said support member to stretch said tissue specimen until the cells thereof are substantially aligned in the direction of stretching movement;

(g) allowing said tissue specimen to rest for at least 24 hours; and

(h) gradually and substantially continuously stretching said support member to stretch said tissue specimen by at least approximatley 0.5% over the course of a 24 hr period while periodically repetitively stretching and releasing said support member to stretch and release said tissue specimen by at least approximately 0.01% for at least a total of 60 seconds during the course of said 24 hr period.

8. In the method of claim 7, said tissue specimen being stretched and released by between 0.01% and 60% during said stretch and release activity.

9. In the method of claim 7, said support member and said tissue specimen being gradually stretched at a rate of between 0.005 mm/hr and 1.0 mm/hr.

10. In the method of claim 7, said support member including a membrane and a pair of spaced upwardly extending support walls on said membrane, said support walls being made of a biocompatible material and having opposed support surfaces thereon which have sufficiently roughened textures to permit attachment of said tissue specimen thereto.

11. In the method of claim 7, said tissue specimen being repetitively stretched and released during activity periods of at least five seconds duration.

12. In the method of claim 11, said activity periods being separated by rest periods of at least five seconds duration during which said tissue specimen is subjected to said gradual and substantially continuous stretch activity but not significant stretch and release activity.