Methods and Devices for Producing Cellular Suspensions from Tissue Samples

a technology of cellular suspension and tissue, which is applied in the direction of biomass after-treatment, instruments, transportation and packaging, etc., can solve the problems of inefficient methods, time-consuming and laborious methods, and insufficient representation of tumor composition

Pending Publication Date: 2021-04-01
BECTON DICKINSON & CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This method efficiently dissociates tissues into viable single-cell suspensions, maintaining cell viability and yield comparable to standard protocols, even without manual mincing or enzyme treatments, facilitating downstream analyses like flow cytometry and nucleic acid sequencing.

Problems solved by technology

The current clinical gold standard for characterizing solid tumors and stratifying patients relies partially on analyzing formalin-fixed paraffin-embedded (FFPE) tumor slices via immunohistochemistry (IHC), but the thin 5-7 micron (μ) sections represent less than 0.01% of the total cell population in a typical tumor biopsy and therefore not a thorough representation of a tumor's composition.
These methods are time-intensive, inefficient, and highly subjective as the skill and experience of the researcher can be a factor.

Method used

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  • Methods and Devices for Producing Cellular Suspensions from Tissue Samples
  • Methods and Devices for Producing Cellular Suspensions from Tissue Samples
  • Methods and Devices for Producing Cellular Suspensions from Tissue Samples

Examples

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example 1

Experimental Design and Controls

[0051]The experiments described in this section were performed using mouse kidney and liver tissue. Each experiment utilized a positive control consisting of a matched piece of organ tissue processed using a Standard Protocol (Table 1). Outputs for evaluating the different tissue dissociation methods tested included assessing (a) the percentage of viable (live) cells in the dissociated sample as a proportion of the total cell output, and (b) the total number of viable cells in the dissociated sample per mg of input tissue. Both measurements were determined using a commercially available Beckman Coulter Vi-Cell cell analyzer (a trypan blue dye exclusion-based assay).

TABLE 1Standard Protocol (Enzyme + Manual Mince)I.Extraction:1.Mice are injected i.p. with 50 / 5 mg / kg of ketamine / xylazine cocktail.2.Once sufficiently sedated, organs are removed for subsequent dissociation.3.The hepatic artery is clipped to ensure euthanization.4.All extracted tissues are...

example 2

[0058]Tissue disruption protocols were performed on mouse liver and kidney tissues as set forth in Table 2 below.

TABLE 2Sample Conditions Example 2SampleOrganProtocol ParametersNo.TypeRAMEnzymeMincingK1KidneyNoneYesYes(Standard protocol)K2KidneyHiYesNoK3KidneyHiNoNoK4KidneyLoNoNoK5KidneyHiYesYesK6KidneyLoYesYesK7KidneyHiNoYesK8KidneyLoNoYesL1LiverNoneYesYes(Standard protocol)L2LiverHiYesNoL3LiverHiNoNoL4LiverLoNoNoL5LiverHiYesYesL6LiverLoYesYesL7LiverHiNoYesL8LiverLoNoYes

[0059]LabRAM I exposure of the tissue samples came after enzyme or PBS (for “No Enzyme” conditions) incubation and prior to initial centrifugation step (Step VI). Conditions labeled “No Mince” skipped the scissor mincing (Step III).

[0060]Hi Ram conditions are as follows: 1 minute exposure to LabRAM I at 90% intensity (100-110 g of acceleration) in an inverted 50 ml conical tube with 50 ml of PBS.

[0061]Lo Ram conditions are as follows: 1 minute exposure to LabRAM I at 50% intensity (62-70 g of acceleration) in an inv...

example 3

[0064]Tissue disruption protocols were performed on mouse liver and kidney tissues as set forth in Table 3 below.

TABLE 3Sample Conditions Example 3SampleOrganProtocol ParametersNo.TypeRAMEnzymeMincingFill (%)K1KidneyNone (StandardYesYesN / Aprotocol)K2KidneyHiLongYesNo100K3KidneyHiLongYesNo50K4KidneyHiShortYesNo100K5KidneyHiShortYesNo50K6KidneyLoLongYesNo100K7KidneyLoLongYesNo50K8KidneyLoShortYesNo100K9KidneyLoShortYesNo50K10KidneyHiLongNoYes100K11KidneyHiLongNoYes50K12KidneyLoLongNoYes100K13KidneyLoLongNoYes50K14KidneyLoLongYesYes100K15KidneyLoLongYesYes50K16KidneyHiLongYesYes100L1LiverNone (StandardYesYesN / Aprotocol)L2LiverHiLongNoNo100L3LiverHiLongNoNo50L4LiverHiShortNoNo100L5LiverHiShortNoNo50L6LiverLoLongNoNo100L7LiverLoLongNoNo50L8LiverLoShortNoNo100L9LiverLoShortNoNo50L10LiverHiLongNoYes100L11LiverHiLongNoYes50L12LiverLoLongNoYes100L13LiverLoLongNoYes50L14LiverLoLongYesYes100L15LiverLoLongYesYes50L16LiverHiLongYesYes100

[0065]Based on the results shown in Study 1 and shown in FI...

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Abstract

Aspects of the present disclosure include methods of producing a cellular suspension from a tissue sample by applying resonant acoustic energy to a container comprising the tissue sample in a manner sufficient to produce a cellular suspension from the tissue sample. Resonant acoustic mixers and kits for use in producing a cellular suspension from a tissue sample are also provided.

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001]Pursuant to 35 U.S.C. § 119(e), this application claims priority to the filing date of U.S. Provisional Patent Application No. 62 / 306,576, filed Mar. 10, 2016; the disclosure of which application is herein incorporated by reference.INTRODUCTION[0002]The current clinical gold standard for characterizing solid tumors and stratifying patients relies partially on analyzing formalin-fixed paraffin-embedded (FFPE) tumor slices via immunohistochemistry (IHC), but the thin 5-7 micron (μ) sections represent less than 0.01% of the total cell population in a typical tumor biopsy and therefore not a thorough representation of a tumor's composition. More specific diagnoses, which will ultimately lead to better patient outcomes, will require clinicians to adopt new, more comprehensive methods of solid tumor analysis.[0003]One of the newest methods on the forefront of cancer microenvironment and solid tumor research involves dissociating solid tumors int...

Claims

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Application Information

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Patent Type & AuthorityApplications(United States)
IPC IPC(8): G01N1/38B01F11/02C12M1/33G01N1/28G01N15/14
CPCG01N1/38B01F11/02C12M45/02G01N1/286G01N2015/1006G01N2001/2866B01F2215/0037B01F2215/0073G01N2001/386G01N15/1459B01F31/80B01F2101/23B01F2101/44C12M45/09C12M47/04C12M27/16
InventorPETTIS, RONALD J.FERGUSON, MITCHELL
OwnerBECTON DICKINSON & CO