SEPARATION METHOD AND DEVICE

TECHNICAL FIELD OF THE INVENTION

The present invention relates to method and a device for separating mononuclear cells from a medium containing red blood cells (erythrocytes), such as a blood sample.

BACKGROUND OF THE INVENTION

The separation of cell containing samples, for example blood, into different fractions by using centrifugation and a density gradient medium has been practised for some time. The principle used is to provide for example a blood sample together with a density gradient medium in a tube and then put the tube into a centrifuge.

The density gradient medium is usually a medium which may form a density gradient upon centrifugation or sedimentation, but it may also be a medium which does not form a density gradient but merely has a different density than the sample medium and forms a step gradient with the sample medium.

The density gradient medium is suitably chosen such that after centrifugation red blood cells, and usually also granulocytes (neutrophils, eosinophils, basophils), are collected at the bottom of the tube below the density gradient medium while the wanted fraction, for example mononuclear cells, MNCs (monocytes and lymphocytes), will stay at the top of the density gradient medium. The plasma will also be separated and stay above the MNCs. The MNCs may be collected in various ways, e.g. by using a pipette. Such a manual process using centrifugation and a density gradient medium is for example described in Boyum, A. Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest. 21, Suppl 97 (Paper IV), 77-89, 1968.

A variety of devices and methods for density gradient-based separation and the recovery of the desired fraction or fractions are described in the prior art, including tubular devices and syringe type devices just to mention a few. Generally, however, the yield of MNCs from density centrifugation devices is usually quite low, 60% or even less.

SUMMARY OF THE INVENTION

It is an object of the present invention is to provide a method and a device which permit the yield of MNCs in density centrifugation to be increased.

This is achieved with a method according to claim 1 and a with a separation device according to claim 5. Suitable embodiments are described in the dependent claims.

DETAILED DESCRIPTION OF THE INVENTION

As mentioned above, the yield of mononuclear cells (MNCs) from density centrifugation of blood is usually relatively low. For some density gradient media like e.g. Ficoll™ a contributory cause is that the gradient density medium induces clotting, i.e. aggregation of red blood cells (erythrocytes). While this increases erythrocyte sedimentation through the Ficoll layer, it is also believed to cause co-aggregation and enclosure of the desired MNCs during the aggregation process, thereby lowering the yield thereof.

According to the present invention, it is proposed to increase the MNC yield by using two different density gradient media, such that the blood sample first comes in contact with a first density gradient medium which at least substantially does not induce or promote aggregation of erythrocytes, and then comes in contact with a second density gradient medium which is capable of inducing or promoting erythrocyte aggregation. In this way, co-aggregation of MNCs may be prevented simultaneously as the sedimentation-increasing effect of the erythrocyte aggregation is benefited from.

Density gradient media which (at least not to any substantial degree) do not induce aggregation of erythrocytes, as well as media which induce aggregation are well-known to a person skilled in the art.

An exemplary density gradient medium that does not aggregate erythrocytes is a medium of Percoll type, including e.g. Percoll™ and Percoll™ PLUS (marketed by GE Healthcare, Uppsala, Sweden). Percoll consists of colloidal silica particles of 15-30 nm diameter which have been coated with polyvinylpyrrolidone. Other examples of non-aggregating density gradient

media include sucrose, and iodinated density gradient media, such as iodixanol, nycodenz and metrizamide.

An exemplary density gradient medium that, on the other hand, aggregates erythrocytes is a medium of Ficoll type, including e.g. Ficoll™, Ficoll™ PM400, Ficoll-Paque™, Ficoll-Paque™ PLUS (marketed by GE Healthcare, Uppsala, Sweden). Ficoll is a neutral, highly branched high- mass, hydrophilic polysaccharide which dissolves readily in aqueous solutions. Another example of aggregating density gradient media is dextran.

Basically, the present invention is applicable to any prior art method and device utilizing a density gradient medium (or media) for separation of MNCs. Exemplary devices include tubular devices, such as tubes and syringe type devices.

The sample medium may be any medium containing desired MNCs in the presence of erythrocytes (in at least interfering amounts) and any other undesired cells. Usually, the sample is human peripheral blood, umbilicial cord blood or placental blood, or optionally animal blood. Also bone marrow samples may be contemplated for use in the present invention.

As mentioned above, the MNCs consist of a mixture of monocytes and lymphocytes, i.e. leucocytes from which granulocytes have been separated and removed. Usually, it is the MNCs content of hematopoietic stem cells or mesenchymal stem cells that are of interest (1-2 % of the MNCs) and can be isolated from the collected MNCs fraction and optionally cultured (mesenchymal stem cells).

When applying the method of the invention to methods and devices using a single density gradient medium, this medium is replaced by two different density gradient media, i.e. a density gradient medium which does not induce erythrocyte aggregation placed on top of a density gradient medium which induces erythrocyte aggregation. The upper density gradient medium should have a density that is at least not higher than the lower density gradient medium.

In case the densities of the two media are the same, or at least essentially the same, the density is chosen such that after centrifugation of the blood sample, the erythrocytes will be separated and positioned below the density gradient medium, while the desired MNCs will collect at the interface between the original blood sample and the upper gradient density medium.

Preferably, however, the two density gradient media have different densities, i.e. the upper density gradient medium has a lower density than the lower gradient density medium. More particularly, the lower gradient density medium should preferably have a density slightly higher than the desired MNCs fraction, whereas the upper density gradient medium should have a density slightly smaller than the MNCs. After centrifugation, the MNCs fraction will then be surrounded by the two density gradient media, collecting at the interface between them.

The two density gradient media may be layered on top of each other in direct contact or optionally separated by a partition means such as a filter, grid or the like which permits passage of liquid and sample components on centrifugation. Such a partition means may also be provided on top of or above the upper density gradient medium layer to prevent mixing thereof with applied sample prior to centrifugation.

Alternatively, the separation device may be compartmented, such as disclosed in our co-pending international application no. PCT/SE2008/000318 entitled "Separation device" (the full disclosure of which is incorporated by reference herein). That separation device has three compartments with operable closing means between the first and second compartments, and between the second and third compartments, respectively. The aggregation-inducing density gradient medium (having a density between the densities of the erythrocytes and the MNCs) is provided in the first (lowermost) compartment, the non-inducing density gradient medium

(having a density between the densities of the MNCs and the blood plasma) is provided in the second (middle) compartment, and the blood sample is applied to the third (uppermost) compartment.

The sizes of the compartments, the amounts of the blood sample, and the densities of the density gradient media are chosen such that the MNCs product after centrifugation ends up in the middle compartment (which is preferably smaller than the first and third compartments such that the purity of the wanted end product can be as high as possible when retrieved from the second compartment, i.e. mixed with a minimum of other constituents). The three compartments are in fluid communication during centrifugation and can be either automatically or manually closed after centrifugation.

In the following Example, the invention is disclosed more specifically for purposes of illustration and not limitation.

EXAMPLE

Materials

Peripheral blood (obtained from Uppsala University hospital, Uppsala, Sweden). Ficoll-Paque™ PLUS (GE Healthcare, Uppsala, Sweden).

Percoll™ (GE Healthcare, Uppsala, Sweden).

Lymphoprep Tubes (50 mL) (Medinor AB, Lidingδ, Sweden); Density gradient medium:

Sodium diatrizoate 9,1 % (w/v); Ficoll polysaccharide 5,7 % (w/v); density 1,077 +/- 0,001 g/mL; Osmolality 290 +/- 15 mOsm. Dulbecco's Phosphate Buffered Saline solution (DPBS) (Cambrex BioSciences, Venders,

France).

CD45 PerCp-Cy5.5 (BD Biosciences, San Jose, CA, USA); monoclonal antibody labelled with

PerCP/Cy5.5 tandem fluorophore.

BD Truecount™ Tubes (BD Biosciences, San Jose, CA, USA). BD FACS Lysing Solution (BD Biosciences, San Jose, CA, USA).

FACS (fluorescence activated cell sorter) (BD Biosciences, San Jose, CA, USA)

Density determinations

To determine dilutions of Ficoll and Percoll that gave comparable densities, Ficoll or Percoll was diluted to 50%, 25% and 12.5%, respectively, with Dulbecco's Phosphate Buffered Saline solution (DPBS). The densities were determined in a Mettler Toledo DE40 Density Meter IP 28054. Dilution and density are linearly correlated, and a concentration of 25% Percoll or 43% Ficoll corresponding to a density of 1.0363 was used in the centrifugation experiment below.

Double density centrifugations

Ten mL of Ficoll or Percoll (diluted to 43% or to 25%, respectively, with DPBS) were added to three Lymphoprep Tubes (50 mL) each, and then 12 mL of blood diluted to 50% with DPBS were carefully layered on top. The three tubes with added Ficoll thus had a layer of Ficoll of density 1.0363 layered on top of the Ficoll layer of density 1.077 already present in the tube, and are below referred to as "Ficoll/Ficoll tubes". Correspondingly, the three tubes with added Percoll had a layer of Percoll with density 1.0363 layered on top of the Ficoll layer of density 1.077 already present in the tube and are below referred to as "Ficoll/Percoll tubes". After sample application, the tubes were documented by photographs. The tubes were then

centrifuged at 400 x g for 20 min at 20 ⁰ C. After centrifugation, the MNC band was aspirated with a Pasteur pipette and diluted to a final volume of 6 mL with DPBS.

FACS analysis of MNCs and red blood cells Ten μL of CD45 PerCp-Cy5.5 antibodies were put as a droplet near the bottom of a BD

Truecount tube. Fifty μL of blood or cells from the MNC fraction were added to the tube and were then mixed for a short while on a table top Vortex mixer. The tubes were incubated in the dark for 15 min at room temperature and then 450 μL of BD Lysing solution diluted 10 fold with water (for the blood sample) or 450 μL DPBS (for the MNC fractions) were added. The tubes were mixed shortly on the table top mixer and incubated for another 15 min in the dark at room temperature.

Cells in the tubes were then counted in the FACS. One setup of gating was used for the red blood cell (RBC) count that yields the percentage of RBC of total cells (sum of RBC, Lymphocytes, Monocytes and Granulocytes). Another setup of gating yielded the count of

Lymphocytes, Monocytes and Granulocytes, and the absolute values were calculated according to the manufacturer's instructions using the standardized amounts of beads in the BD Truecount tubes.

Calculation of yield in the MNC fraction:

MNCs are the sum of Lymphocytes and Monocytes.

Yield (in %) of MNCs = # of MNCs in the fraction x 100 # of MNCs in the blood

Calculation of purity in the MNC fraction:

Purity (%) of MNCs = # of MNCs in the fraction x 100 # MNCs plus Granulocytes in the fraction

Results

As documented on photographs, the Ficoll/Ficoll tubes showed a substantial aggregation of red blood cells already before centrifugation compared to the Ficoll/Percoll tubes.

After centrifugation there was also a difference in the pellet. The Ficoll/Ficoll tubes showed a compact pellet, whereas the Ficoll/Percoll tubes showed a more diffuse pellet with some less clear solution under the MNC band, indicating that an increased centrifugation time for the

Ficoll/Percoll tube would give a more compact pellet with more clear solution under the MNC band.

The MNC fractions were analyzed for the yield of MNCs. The results are presented in Table I below.

Table I. Yield of MNCs for the different tubes

The mean yield for Ficoll/Ficoll tubes was 32% and the mean yield for Ficoll/Percoll tubes was 39%. Thus, by making the blood sample first contact a density gradient medium (Percoll) that does not cause aggregation of the blood cells, and subsequently a density gradient medium which causes aggregation, the yield was increased.

It is to be understood that the invention is not limited to the particular embodiments of the invention described above, but the scope of the invention will be established by the appended claims.