In vitro homogenous cell block, method of making and using

Abstract

A solid composition comprising a homogenous cell block able to be used as a positive control for biomarkers in immunohistochemistry experiments, such as slide scanning and image analysis. The homogenous cell block is produced using a three layered vertical apparatus to create an evenly distributed suspension of FFPE cells, wherein the cells are mixed with 3% agarose while still rotating within the apparatus's middle layer. The injection of the cell mixture into a mold creates a homogeneous cell block where each cell, or ratio of different types of cells, is evenly distributed. The cell mixture within the cell block may further comprise: a mixture of the same type of cell with different genetic modifications; a mixture of the same type of cell with different protein or nucleic acids expression; and a mixture of different types of cells with different genetic backgrounds, and/or different expression level of genes and/or proteins.

Claims

1. A positive control, comprising a solid composition of a homogenous mixture of cells within a formalin fixed paraffin embedded block, wherein said mixture of cells are evenly distributed throughout said solid composition's volume with a uniform distribution and density of non-clumped cells of a same cell type, or of a ratio of different cell types, within the mixture of cells, and the cells of each type are of a pre-determined number; wherein the positive control is able to function as a biomarker in an immunohistochemistry experiment to detect and diagnose abnormal medical conditions in a patient; and wherein the composition's uniform distribution and density of non-clumped cells is achieved by adjusting the size of a center hole in a vertical, rotating apparatus to pass the cells of the pre-determined number downward through the hole when preparing the composition.

2. The solid composition of claim 1, wherein said mixture of cells further comprises a pre-determined ratio of: a different types of cells; a different combination of a certain percentages of each cell type; and/or a same type of cells exposed to different treatment protocols.

3. The solid composition of claim 2, wherein the pre-determined ratio and/or a quantity of the different cells within said mixture of cells are able to be verified within a cross-section of said solid composition by cell counting, and/or by DNA extraction and quantification.

4. The solid composition of claim 1, wherein said mixture of cells comprises a pre-determined ratio of: a same type of cells with different genetic modifications; a same type of cells with different protein or nucleic acids expression; a different types of cells with different genetic backgrounds; and/or a different types of cells with different protein or nucleic acid expression levels.

5. The solid composition of claim 4, wherein the pre-determined ratio and/or a quantity of the different cells within said mixture of cells are able to be verified within each cross-section of said solid composition by cell counting, and/or by DNA extraction and quantification.

6. The solid composition of claim 1, wherein said mixture of cells comprises a pre-determined ratio of different types of cells.

7. The solid composition of claim 2, wherein said mixture of cells comprises the different combination of the certain percentages of each cell type.

8. The solid composition of claim 2, wherein said mixture of cells comprises the same type of cells exposed to different treatment protocols.

9. The solid composition of claim 4, wherein said mixture of cells comprises the same type of cells with different genetic modifications.

10. The solid composition of claim 4, wherein said mixture of cells comprises the ratio of the same type of cells with different protein or nucleic acids expression.

11. The solid composition of claim 4, wherein said mixture of cells comprises the different types of cells with a different genetic background.

12. The solid composition of claim 4, wherein said mixture of cells comprises the different types of cells with different protein or nucleic acid expression levels.

13. The solid composition of claim 1, wherein said solid composition comprises a plurality of 5 m cross-sections, each section able to function as a positive control for in vitro immunohistochemistry experiments with an even distribution and density of non-clumped cells throughout the cross-sections.

14. The solid composition of claim 1, wherein said solid composition comprises agarose evenly distributed throughout the composition at a uniform concentration.

15. The solid composition of claim 14, wherein said solid composition comprises 3% agarose.

16. The positive control of claim 1, wherein the solid composition comprises a cylindrical shape containing 1000 to 1200 cross-sectional slices of 5 m thickness per slice.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) The in vitro homogenous cell block, methods of making, and methods of use that embody the above and other inventive features will now be described with reference to the following drawings:

(2) FIG. 1 is a flowchart of steps for preparing in vitro homogenous cell blocks.

(3) FIG. 2 is an image of an apparatus used in steps of the method of preparing the homogenous cell blocks.

(4) FIG. 3 is a table comprising the cell block paraffin processing schedule.

(5) FIG. 4 is a table comprising the Hematoxylin Staining Procedure.

(6) FIG. 5 is a table comprising the DNA yield of different cell blocks of an exemplification utilizing the present method.

(7) FIG. 6 illustrates images of the exemplification of FIG. 5 demonstrating the homogenous nature of the cell distribution.

DETAILED DESCRIPTION

(8) The following is a method of the present invention to prepare 10 m FFPE sections of homogenous cell blocks, although it is noted that one of skill in the art would readily know how to adjust this disclosure for producing other types of homogenous cell blocks, such as 5 m FFPE sections.

(9) Step (120) Comprises the Pre-Treatment of Cells for Cell Block Preparation:

(10) 1. Remove tissue culture medium and wash cells once with phosphate buffered saline (PBS).

(11) 2. Detach cells using trypsin and stop trypsinization by adding culture medium.

(12) 3. Centrifuge cells at 800g for 5 minutes and remove supernatant.

(13) 4. Wash cell pellet once with PBS.

(14) 5. Re-suspend the cells in 15 ml PBS and place them in the upper chamber of the apparatus of FIG. 2.

(15) 6. Pass the cell suspension through a cell strainer in the upper chamber of FIG. 2 (e.g. 70 micrometer).

(16) 7. Perform cell counting and cell viability using a Cellometer Auto T4 and Trypan blue staining.

(17) 8. Fix the cells for 24 hours at room temperature with freshly prepared 2% paraformaldehyde (PFA) in PBS.

(18) 9. After fixation, centrifuge the cells at 800g for 5 minutes and remove the supernatant.

(19) 10. Rinse the cell pellet once with 70% ethanol. Keep at 4 C. until use.

(20) Step (140) Comprises Cell Block Preparation

(21) Cylindrical molds of size (4 mm in radius, 145 mm in length) (MOLD A) are used for making the cell block. The one time use cell block Mold A's (first molds) are kept at 10 C. for one hour before use. The cell pellet is re-suspended in PBS.

(22) Because cell pellets fixed in formalin or paraformaldehyde (PFA) become very tough when the pellets are subsequently embedded into paraffin, they create a pellet with very dense cells. To prevent this, the fixed cell pellet is passed through the apparatus 200 illustrated in FIG. 2, which mixes the cells with the solution simultaneously while it goes through the upper chamber to the middle chamber. Therefore, this makes them into a very even suspension of cells.

(23) As shown in apparatus 200 of FIG. 2, the re-suspended cells are passed through the upper chamber 210 through the hole in the middle chamber 220. The secondary hole or tube 250 then injects a fluid simultaneously (e.g. 3% agarose) onto the rotating hole to mix with the cells. The cell/agarose mixture then flows through the middle chamber hole 240 onto the lower collection chamber 230.

(24) The cell/agarose mixture is then injected into the Mold A, which is subsequently kept at 10 C. for 5 minutes. The solid composition (i.e. cell block) is then removed from the Mold A and put into individual 50 ml tubes containing 70% ethanol. The cell blocks are processed for paraffin on individual 50 ml tubes using the sequential steps of procedures listed in the table of FIG. 3 comprising the Cell Block Paraffin Processing Schedule.

(25) Immediately after paraffin processing, individual blocks are removed from the paraffin and embedded onto a cubed paraffin Mold B (2 cm*2 cm*2 cm) (second mold). The cell blocks are wrapped with parafilm and kept in air-tight box at 4 C. until sectioning.

(26) Step (160) Comprising Sectioning Cell Blocks and Hematoxylin Staining

(27) The cell blocks from Mold B are removed from the 4 C. refrigerator and mounted onto cassettes for sectioning utilizing a Leica microtome. A single-use high profile microtome blade is used for the sectioning of each cell block; 10 m FFPE sections are prepared from each block and sections are mounted onto positively charged glass slides and left for air drying at room temperature for 30 minutes. The slides are then baked in a 56 C. oven for 20 minutes before hematoxylin staining using the procedure shown in the table of FIG. 4.

(28) Confirm Cell Block Homogeneity

(29) To confirm homogeneity of the cell blocks, multiple sections (e.g. 1000 cross-sections) are cut from a cell block containing millions of cells, and DNA extraction is performed on selected individual sections (e.g. every 100th section) as per step 180, and the DNA is quantified as per step 200. Additionally, digital microscopy images taken of hematoxylin-stained cell sections show the cells scattered evenly within the cell block section.

(30) Step (180) Comprises DNA Extraction from 10 m FFPE Cell Block Section

(31) DNA extraction methods are well known in the art. By way of exemplification, DNA extraction may be performed using the Qiagen DNeasy Blood & Tissue Kit (cat. #69504, Qiagen, USA) according to the manufacturer's protocol with two exceptions: 1) Proteinase K digestion is performed overnight in a 56 C. water bath; and, 2) DNA is eluted with 100 l elution buffer twice to yield a total volume of 200 l DNA.

(32) Step (200) Comprises DNA Quantitation Using PicoGreen Fluorescent Dye

(33) Likewise, DNA quantification methods are well known in the art. By way of exemplification, the DNA is quantified using a Quant-iT PicoGreen dsDNA Reagent and Kit (Molecular Probes, Eugene, Oreg.). Standard curve samples are freshly diluted from the Lambda DNA standard before each batch of sample measurements. DNA measurement of unknown samples are performed according to the manufacturer's recommendation. Fluorescence readings are taken with a TBS-380 Mini-Fluorometer (Turner Biosystems, Sunnyvale, Calif.) using 1010 mm square polystyrene disposable cuvettes. Sample DNA concentrations are then extrapolated from the Lambda DNA standard curve.

EXEMPLIFICATION

(34) An in vitro homogenous cell block comprising HCT-116 cells, wherein the block is produced by the method of the present invention as disclosed in FIGS. 1 & 2, was quantified for the amount of DNA yield in a 10 micrometer thick slice of the block. The results are shown in the table of FIG. 5 disclosing: the cell block initial number of cells (20, 40, 80 and 160 million); the cell number per 10 micrometer section from 1200 sections total (11,650; 23,889; 61,170; and 126,500) respectively; and the DNA yield per the corresponding 10 micrometer section (62, 131, 245, and 512).

(35) Images generated using Aperio ScanScope whole slide scanner of the above hematoxylin stained blocks of cells of 40 million (Section A) and 80 million (Section B) demonstrate the homogenous nature of the cells blocks (See FIG. 6). Nuclear cell count on hematoxylin positive cell section was performed using Aperio ImagesScope program. The left image (Section A) shows 23,000+ cells; and the right image (Section B) shows 61,000+ cells, both of which comprise the cells evenly distributed throughout the section.

(36) While the present invention has been described with reference to a few specific embodiments, the description is illustrative of the invention and is not to be construed as limiting the invention. Various modifications may occur to those skilled in the art without departing from the true spirit and scope of the invention as defined by the appended claims.