Low pressure, shear-type cell homogenizer and method of using

• US 5,390,859 A
• Filed: 10/06/1993
• Issued: 02/21/1995
• Est. Priority Date: 10/06/1993
• Status: Expired due to Fees

• Current Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Original Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Inventors: Rajasekaran, Ayyappan K.
• Primary Examiner(s): Eley, Timothy V.
• Application Number:
  US08/132,475
• Time in Patent Office: 503 Days
• Field of Search: 241/2, 241/114, 241/169, 241/169.2, 241/169.1, 241/199, 241/199.7, 241/199.8, 241/199.9, 241/199.11, 241/199.12, 241/261.2, 241/257.1, 241/259
• US Class Current: 241/2
• CPC Class Codes: B02C 19/08 Pestle and mortar
• CPC Class Codes: C12M 47/06 Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material lysis of microorganisms C12N1/06; extracting or separating nucleic acids from biological samples C12N15/1003
• CPC Class Codes: C12N 1/005 after treatment of microbial biomass not covered by C12N1/02 - C12N1/08

Abstract

A method for rupturing cell walls and membranes of organic cells while leaving intra-cellular organelles substantially intact employs a culture dish for holding the cells. A pestle includes a cylindrical portion that metes with the culture dish. Extending from the cylindrical portion is an axially aligned shaft. The method includes the steps of: emplacing a layer of cells in the culture dish; placing the pestle in contact with the layer of cells; applying a pressure axially to the shaft to force the pestle into contact with the cells; and rotating the shaft and pestle a plurality of reverse-direction partial rotations to achieve a rupture of cellular membranes of cells within the culture dish. Both the base of the culture dish and the pestle are substantially planar thereby enabling uniform pressure application across the base of the culture dish.

Claims

1. A method for homogenizing cells while leaving substantially intact, intracellular organelles, the method employing a flat-bottom dish for holding the cells, a pestle having a planar bearing member that intimately mates with said flat-bottom dish, the method comprising the steps of:

emplacing said cells and an isotonic solution in said dish;

placing said bearing member of said pestle in contact with said cells;

applying pressure between said bearing member and said dish, said pressure substantially evenly distributed over mating surfaces of said bearing member and said dish, and

causing relative rotative movement between said bearing member and said dish, said relative rotative movement comprising plural successive clockwise and counterclockwise rotations.

2. The method as recited in claim 1 wherein said pressure does not exceed ten pounds and each rotation does not exceed 180°

.

3. The method as recited in claim 1 wherein said relative pressure is within the range of two to four pounds.

4. The method as recited in claim 1 wherein each rotation is approximately 90°

and a total number of said successive rotations does not exceed 4.

5. Apparatus for homogenizing cells while leaving intra-cellular organelles substantially intact, said apparatus comprising:

a cell dish having a recessed, continuous planar bottom surface for receiving and holding cells to be homogenized;

a pestle having a single planar bearing member that intimately mates with said recessed planar bottom surface

of said cell dish; and

means for applying a relative pressure between said single planar bearing member and said recessed planar bottom surface so that said relative pressure is substantially evenly distributed across mating surfaces therebetween and is of a magnitude to rupture cell walls while leaving intra cellular organelles intact, and for further causing plural relative counter-rotations between said recessed planar bottom surface and said single planar bearing member.

6. The apparatus as recited in claim 5 wherein said single planar bearing member is roughened.

7. Apparatus for homogenizing cells while leaving intra-cellular organelles substantially intact, said apparatus comprising:

a cell dish having a planar bottom surface for receiving cells to be homogenized;

a first pestle having a planar bearing surface that mates with said planar bottom surface of said cell dish, said first pestle having a central orifice;

a second pestle having a planar bearing surface that mates with said planar surface of said cell dish, fitted within said second orifice, said first and second pestles independently movable with respect to each other; and

means for applying a relative pressure between said pestles and said planar bottom surface that is substantially evenly distributed across said planar bottom surface and for causing relative rotations between said pestles and said planar bottom surface.

8. The apparatus as recited in claim 7 wherein said planar bearing surfaces are roughened.