Apparatus and method for separating microscopic units in a substantially continuous density gradient solution

• US 5,316,540 A
• Filed: 01/13/1993
• Issued: 05/31/1994
• Est. Priority Date: 01/13/1993
• Status: Expired due to Term

• Current Assignee: Gambro, Inc. (DaVita Incorporated)
• Original Assignee: Cobe Cardiovascular Incorporated (LivaNova Plc)
• Inventors: McMannis, John D., London, Nicholas
• Primary Examiner(s): JENKINS, ROBERT
• Application Number:
  US08/003,559
• Time in Patent Office: 503 Days
• Field of Search: 494/37, 494/45, 494/22, 494/23, 494/27, 494/28, 494/31, 494/35, 422/72, 604/4, 604/5, 604/6, 210/781, 210/782
• US Class Current: 494/37
• CPC Class Codes: A61M 5/1483 using flexible bags externally pressurised by fluid pressure
• CPC Class Codes: B04B 5/0428 with flexible receptacles
• CPC Class Codes: B04B 5/0442 with means for adding or withdrawing liquid substances during the centrifugation, e.g. continuous centrifugation
• CPC Class Codes: G01N 15/042 by centrifuging and investigating centrifugates centrifuges per se B04B
• CPC Class Codes: G01N 15/05 in blood

Abstract

An apparatus and method for the separation of a predetermined type of microscopic unit (e.g., islets of Langerhans) from a given medium (e.g., pancreatic digest) by centrifuging in a substantially continuous density gradient. In one embodiment, a substantially continuous density gradient solution is provided to a flexible processing bag appropriately positioned within an upper chamber of a rotating variable volume centrifuge. The solution having a plurality of the desired microscopic units therein is loaded onto the gradient and such units migrate to a particular radial position within the gradient based upon the density of such units. Once appropriately separated by the formation of regions, a fluid is provided to a lower chamber of the centrifuge bowl during continued rotation thereof to sequentially remove fractions from the processing bag from an inward location to an outward location, at least one of which fractions includes the band of the desired microscopic units. The desired microscopic units of a given fraction(s) may therefore be provided to an appropriate collector.

Claims

1. A method for separating a plurality of at least one type of an microscopic unit from an microscopic unit-containing fluid, comprising the steps of:

providing a centrifuge;

positioning a processing container in said centrifuge;

rotating said centrifuge and said processing container;

providing a substantially continuous density gradient solution to said processing container;

providing said microscopic unit-containing fluid to said processing container;

separating said plurality of microscopic units from remaining portions of said microscopic unit-containing fluid based upon a density of said microscopic units within said substantially continuous density gradient solution; and

harvesting said plurality of separated microscopic units from said processing container during at least a portion of said rotating step.

2. A method, as claimed in claim 1, wherein:

said providing said substantially continuous density gradient solution step is performed during at least a portion of said rotating step.

3. A method, as claimed in claim 1, wherein:

said substantially continuous density gradient solution extends radially within said processing container relative to said rotational axis.

4. A method, as claimed in claim 3, further comprising:

maintaining said radial extent of said substantially continuous density gradient during said harvesting step.

5. A method, as claimed in claim 1, wherein:

said providing said microscopic unit-containing fluid step is performed after said providing a substantially continuous density gradient solution step.

6. A method, as claimed in claim 1, wherein:

said providing said microscopic unit-containing fluid step is performed before said providing a substantially continuous density gradient solution step.

7. A method, as claimed in claim 1, wherein:

said separating step comprises creating a plurality of regions, each said region being defined by at least one density, said regions collectively extending from a first position within said processing container to a second position within said processing container radially outward from said first position, said plurality of separated microscopic units being contained within at least one of said regions.

8. A method, as claimed in claim 7, wherein:

said harvesting step comprising sequentially removing said regions from said processing container in an order of increasing density.

9. A method, as claimed in claim 1, wherein:

said microscopic units are islets of Langerhans.

10. A method for using a centrifuge for separating a plurality of at least one type of a microscopic unit having a density from a microscopic unit-containing fluid, said centrifuge having a container that is centrifugally rotated by the centrifuge and being adapted to feed fluids into the container while the container is rotating and being further adapted to remove fluids from the container while the container is rotating, comprising the steps of:

centrifugally rotating the container;

creating a continuous density gradient fluid having a maximum density that is not less than the density of the microscopic unit and a minimum density that is not more than the density of the microscopic unit and further having a substantially continuous range of densities between the maximum density and the minimum density;

feeding the continuous density gradient fluid into the container while the container is rotating with the density of the fluid being fed gradually decreasing from the maximum density of the continuous density gradient fluid to the minimum density of the continuous density gradient fluid;

feeding the microscopic unit containing fluid into the container while the container is rotating;

continuing to centrifugally rotate the container to concentrate the microscopic units in a fraction of the continuous density gradient fluid that has a density similar to the density of the microscopic units; and

removing from the container while the container is rotating the fraction of the continuous density gradient fluid having a density that is similar to the density of the microscopic units and concurrently removing the concentrated microscopic units from the container.

11. A method as defined in claim 10 wherein the step of feeding the continuous density gradient fluid is performed before the step of feeding the microscopic unit containing fluid.

12. A method as defined in claim 10 wherein the step of feeding the microscopic unit containing fluid is performed before the step of feeding the continuous density gradient fluid.

13. A method as defined in claim 10 further comprising the step of:

removing from the container while the container is rotating a fraction of the continuous density gradient fluid having a density less than the density of the microscopic units.

14. A method as defined in claim 10 further comprising the step of:

removing from the container while the container is rotating a fraction of the continuous density gradient fluid having a density greater than the density of the microscopic units.

15. A method as defined in claim 10 wherein:

the density of each one of the plurality of microscopic units is less than a higher microscopic unit density and more than a lower microscopic unit density; and

the fraction of the continuous density gradient fluid has a substantially continuous density from approximately the lower microscopic unit density to approximately the higher microscopic unit density.

16. A method, as claimed in claim 10, wherein:

said microscopic units are islets of Langerhans.