Method of growing a confluent layer of cells on a porous or semi-permeable substrate and apparatus for practicing the method

• US 4,608,342 A
• Filed: 05/14/1984
• Issued: 08/26/1986
• Est. Priority Date: 05/13/1983
• Status: Expired due to Fees

• Current Assignee: Nees, Stephan
• Original Assignee: Nees, Stephan
• Inventors: Nees, Stephan
• Primary Examiner(s): Shapiro, Lionel M.
• Assistant Examiner(s): PARR, MARGARET M
• Application Number:
  US06/609,841
• Time in Patent Office: 834 Days
• Field of Search: 435/283, 435/284, 435/287, 435/297-300, 435/301, 435/310, 435/240, 435/285, 435/286, 435/243, 435/261, 435/30, 435/31, 435/311
• US Class Current: 435/401
• CPC Class Codes: C12M 23/22 Transparent or translucent parts glassware for laboratory use B01L3/00
• CPC Class Codes: C12M 25/04 in combination with well or multiwell plates, i.e. culture inserts

Abstract

A method and apparatus are disclosed for growing a confluent layer of cells on the surface of a substrate which is held by a holding and positioning device so as to be in the form of a taut, planar substrate having upper and lower exposed surfaces. The device is arranged in a nutrient-containing receptacle which also contains a plate material having a solid upper planar surface extending above the plane of the bottom of the receptacle. The lower layer of the receptacle is in contact with the upper surface of the plate material and cells disposed on the upper surface of the substrate are found to anchor to the substrate and grow to a confluent layer.

Claims

1. A method for growing a confluent layer of cells on a substrate surface, comprising the steps of:

providing a porous or semi-permeable substrate in the form of a tensioned, taut, planar substrate having exposed upper and lower surfaces and independently held in such configuration by a tensioning and holding device;

providing a receptacle containing fluid nutrient medium for cell growth, said receptacle having a bottom surface and upstanding side walls about the periphery thereof;

arranging within said receptacle a body of solid, plate material having a bottom surface in contact with the bottom surface of said receptacle and an upper solid, planar surface extending above the plane of the bottom surface of said receptacle;

arranging said holding and tensioning device within said receptacle such that the lower surface of said substrate is in contact with the upper non-porous surface of said plate material and such that the substrate is surrounded by the fluid nutrient medium for cell growth;

providing on the upper surface of said substrate a source of cells; and

maintaining said arrangement under conditions, and for a time, effective to produce a confluent layer of cells on the upper surface of said substrate, whereby a region is created created between the mutually contacting surfaces of said substrate and said plate material and in said region substances synthesized by the cells in aid of their anchoring to said substrate and passing through the porous or semi-permeable substrate are enriched proximate to the cells and not substantially decomposed, modified or diluted by the surrounding nutrient medium.

2. The method according to claim 1 wherein said plate material consists of a transparent glass body.

3. The method according to claims 1 or 2 wherein said substrate is a porous filter composed of a material selected from the group consisting of cellulose acetate and polycarbonate.

4. The method according to claims 1 or 2 wherein said substrate has a porosity of less than or equal to 0.5 microns.

5. The method according to claims 1 or 2 wherein said substrate comprises a dialysis membrane.

6. A method for growing a confluent layer of cells on a substrate, comprising:

providing a porous or semi-permeable substrate in the form of a tensioned, taut, planar substrate held about its periphery by a holding and tensioning device, said held substrate having exposed upper and lower surfaces;

placing said substrate on a body of solid, plate material such that the lower surface of said substrate is in contact with an upper solid planar surface of said body of plate material;

depositing a source of cells on the upper layer of said substrate;

providing a source of fluid nutrient medium for cellular growth surrounding said substrate; and

maintaining said arrangement under conditions, and for a time, effective to produce a confluent layers of cells on the upper surface of said substrate, whereby a region is created between the mutually contacting surfaces of said substrate and said plate material, and in said region substances synthesized by the cells in aid of their anchoring to said substrate and passing through the porous or semi-permeable substrate are enriched proximate to the cells and not substantially decomposed, modified or diluted by the surrounding nutrient medium.

7. An apparatus for growing a confluent layer of cells on a substrate surface, comprising:

a receptacle adapted to receive a fluid nutrient medium, having a bottom surface and upstanding side walls about the periphery thereof;

a body of solid, plate material arranged within said receptacle and having a bottom surface in contact with the bottom surface of said receptacle and an upper solid planar surface extending above the plane of the bottom surface of said receptacle; and

a taut, planar porous or semi-permeable substrate having exposed upper and lower surfaces and held in said taut, planar position by a tensioning and holding device, said tensioning and holding device being arranged within said receptacle such that the lower surface of said substrate is in contact with the upper solid planar surface of said plate material and such that said substrate is adapted to be surrounded by fluid nutrient medium in said receptacle, whereby a region is created between the mutually contacting surfaces of said plate material and said substrate in which substances synthesized by cells in aid of their anchoring to said substrate and passing through said substrate are enriched proximate to the cells and not substantially decomposed, modified or diluted by surrounding nutrient medium, the upper surface of said substrate being adapted to receive a source of cells thereon.

8. The apparatus according to claim 7 wherein said substrate and holding and tensioning device comprises first and second ring members, said second ring member having along its inner edge a recess adapted to receive said first ring member and the peripheral area of said substrate disposed between said first and second ring members.

9. The apparatus according to claim 8 wherein said ring members are circular in shape.

10. The apparatus according to claims 7, 8 or 9 wherein said recess in said second ring member is dimensioned such that the recess covers about one-fourth of the cross-sectional area of said second ring member.

11. The apparatus according to claims 7, 8 or 9 wherein said ring members are circular in shape, said plate material is circular in shape, and wherein the inner diameter of said ring members is slightly greater than the outer diameter of said plate material.

12. The apparatus according to claims 7, 8 or 9 wherein said plate material is composed of glass.

13. A method for constructing an apparatus for growing a confluent layer of cells on a substrate comprising the steps of:

providing a first ring member;

placing said first ring member on a base plate in a manner such that a raised portion of said base plate fills the space enclosed by said first ring member and the surface of said raised portion facing away from the base plate lies in the same plane as that of the upper surface of said first ring;

arranging across the upper surface of said combined first ring and base plate a planar porous or semi-permeable substrate having upper and lower surfaces;

providing a second ring member having a recess along its inner edge;

urging said second ring member onto the upper surface of said first ring member to clamp said first ring member and edge portions of said substrate in said recess;

removing said clamped first and second rings and the substrate therebetween from said base plate;

providing a receptacle containing fluid nutrient medium for cells and arranging within said receptacle a plate material having an upper solid planar surface extending above the plane of the bottom surface of said receptacle; and

disposing said clamped first and second rings and the substrate therebetween onto the upper surface of said plate material such that the bottom planar surface of said substrate is in contact with said upper surface of said plate material, and such that said substrate is surrounded by said nutrient medium, whereby a region is created between the mutually contacting surfaces of said plate material and said substrate in which substances synthesized by cells in aid of their anchoring to said substrate and passing through said substrate are enriched proximate to the cells and not substantially decomposed, modified or diluted by surrounding nutrient medium.