WO2024099865A1

WO2024099865A1 - Cell suspension for use in the treatment of stroke patients

Info

Publication number: WO2024099865A1
Application number: PCT/EP2023/080544
Authority: WO
Inventors: Francisco MONICHE ALVAREZ; Joan Montaner Villalonga; Juan Antonio CABEZAS RODRÍGUEZ; Alejandro GONZÁLEZ GARCÍA; Manuel MEDINA RODRIGUEZ
Original assignee: Servicio Andaluz De Salud
Priority date: 2022-11-08
Filing date: 2023-11-02
Publication date: 2024-05-16

Abstract

The present invention refers to a cell suspension of autologous adult bone marrow-derived mononuclear cells for use in the treatment or amelioration of a subject suffering or having suffered from an ischemic or a hemorrhagic stroke and methods of obtaining such cell suspension.

Description

CELL SUSPENSION FOR USE IN THE TREATMENT OF STROKE PATIENTS

TECHNICAL FIELD

The present invention relates to a cell suspension of adult bone marrow derived cells which can be used in the treatment or amelioration of ischemic or hemorrhagic stroke patients.

BACKGROUND OF THE INVENTION

Stroke is one the leading causes of morbidity and long-term disability in the world, with 15% to 30% of survivors being permanently disabled.¹ In the era of thrombectomy, acute stroke management has faced a revolution, improving long-term prognosis in those patients treated with recanalization therapy. Only intravenous thrombolysis, mechanical thrombectomy, and the stroke unit care have demonstrated to improve stroke outcomes. However, even in those patients treated with mechanical thrombectomy, near half of the patients remains with disability at 3 months. To date, no effective neuroprotective or neurorestorative therapies have been approved for ischemic stroke.² Therefore, new approaches to improve those patients with moderate-to-severe neurological deficits due to established stroke with high rate of long-term disability are urgently needed.

Cell-based therapy is a potential approach in the treatment of ischemic stroke.³ Bone marrow mononuclear cells (BM-MNCs) are a fraction of bone marrow that contain a population of mesenchymal and hematopoietic stem cells. Autologous BM-MNCs transplantation has demonstrated efficacy in stroke animal models.⁴'⁵ Several biological effects, such as attenuation of neuronal death, modulating microglia, reducing proinflammatory responses, increasing neoangiogenesis, and promoting proliferation of endogenous neural stem cells have been invoked.^{6 7} Autologous BM-MNCs can be rapidly obtained within hours, making them an attractive candidate for promoting stroke recovery even in the acute phase. The inventors and others have shown the safety and feasibility in pilot clinical trials in the acute phase of stroke.⁸'⁹ In a meta-analysis of stem cell-based therapies, administration of cell therapy in the acute phase of stroke seems to have higher efficacy than later windows.¹⁰ Also, there is preliminary evidence of a dose-response curve, with higher number of cells administered being more efficacious. However, no previous randomized clinical trial has tested the efficacy of different doses of intraarterial BM-MNCs in acute stroke patients. The inventors have therefore assessed in clinical trial NCT02178657 the safety and efficacy of autologous BM-MNCs transplantation in a multicentre single-blind (outcomes assessor) phase lib controlled clinical trial with two different doses of BM-MNCs in acute patients with middle cerebral artery (MCA) ischemic stroke¹¹. Whilst the clinical trial protocol has been described, the final results as well as the composition of the autologous BM-MNCs have not been previously disclosed.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1. Clinical trial flow chart. *One patient randomized to the 5x106 BM-MNC group, finally received 2x106 BM-MNCs, so was analyzed in the low-dose group in the per-protocol analysis.

Fig. 2. Outcomes at 6 months according to the scores for the modified Rankin Scale in the intention-to-treat population.

Fig. 3. Effects of BM-MNCs on clinical measures in primary and secondary outcomes in the intention-to-treat population. Data are n/N (%), n (%), or median (IQR), unless otherwise indicated. Intervention effects on outcomes were examined by general linear models or binary logistic regression, which were adjusted for baseline NIHSS and thrombectomy. NIHSS=National Institutes of Health Stroke Scale. OR=odds ratio. mRS=modified Rankin scale. *odds ratio indicating the odds of improvement of 1 point on the mRS.

Fig. 4. Effects of BM-MNCs on clinical measures in primary and secondary outcomes in the per- protocol population. Data are n/N (%), n (%), or median (IQR), unless otherwise indicated. Intervention effects on outcomes were examined by general linear models or binary logistic regression, which were adjusted for baseline NIHSS and thrombectomy. NIHSS=National Institutes of Health Stroke Scale. OR=odds ratio. mRS=modified Rankin scale. *odds ratio indicating the odds of improvement of 1 point on the mRS.

Fig.5. Infarct lesion topography. Left side shows the control patients group and rights side the patient group treated with BM-MNCs according to present invention. Fig.6. Variations in fractional anisotropy values from baseline to 6 months of follow-up. The white matter tracts in brain that have statistically significant better axonal integrity measured by fractional anisotropy are shown in the figure.

BRIEF DESCRIPTION OF THE INVENTION

The problem addressed by the present invention is to provide patients that have suffered an acute ischemic stroke with an improved therapeutic alternative. Such therapeutic improved alternative is provided by the present invention in the form of a cell suspension of autologous adult bone marrow derived cells as defined by the aspects and preferred embodiments referred to below.

SUMMARY OF THE INVENTION

In one aspect the present invention relates to a cell suspension comprising from 1 x 10⁷to 8 x 10⁹ of autologous mononuclear cells derived from the bone marrow of a human subject, wherein of the total number of mononuclear cells in said cell suspension i. 1.06% to 20.27% are hematopoietic stem cells that express CD34; ii. 5.64% to 30.63% express CD31; iii. 0.3% to 1.81% are progenitor cells that express CD133; iv. 0.42% to 9.95% express CD146; v. 2.71% to 71.68% are CXCR4+ cells and 0% to 2.19% are VEFGR2+ cells vi. 0% to 1,14% are CD133-/CD31+/CD146+ circulating endothelial cells (CECs) for use in the treatment or amelioration of a subject suffering or having suffered from an ischemic stroke or a hemorrhagic stroke.

In one embodiment of the cell suspension for the use according to present invention the cell suspension comprises 2 x 10⁷ to 4 x 10⁹ mononuclear cells, preferably 5 x 10⁷ to 2 x 10⁹, more preferred 8 x 10⁷to 1 x 10⁹, even more preferred 9 x 10⁷to 9 x 10⁸, most preferred 1 x 10⁸ to 8.1 x 10⁸ mononuclear cells.

In a further embodiment of the cell suspension for the use according to present invention the cell suspension further comprises a lactated Ringer solution, wherein preferably the lactated Ringer solution further comprises about 1% albumin and/or about 2.5% glucose. In one embodiment of the cell suspension for the use according to present invention the ischemic stroke is selected from a middle cerebral artery (MCA) ischemic stroke and ischemic lesions in other regions of the brain, such as for example the anterior cerebral artery (ACA) or the posterior cerebral artery (PCA). The ischemic stroke may therefore be selected from a middle cerebral artery (MCA) ischemic stroke, a anterior cerebral artery (ACA) stroke or a posterior cerebral artery (PCA) stroke. In a preferred embodiment the ischemic stroke is an acute ischemic stroke.

In one embodiment of the cell suspension for the use according to present invention the hemorrhagic stroke is selected from lobar hemorrhagic stroke or deep hemorrhagic stroke. In a preferred embodiment the hemorrhagic stroke is an acute hemorrhagic stroke.

In one aspect of the embodiments of present invention the cell suspension is administered into the stroke related area via intra-arterial administration. In a preferred embodiment the cell suspension is administered into the middle cerebral artery (MCA), the anterior cerebral artery (ACA), or the posterior cerebral artery (PCA).

In one embodiment the intra-arterial administration of the cell suspension is performed at a rate of between 0.2ml - 1.5ml/min, preferably of between 0.5ml-lml/min into the stroke related area.

In a further aspect of present invention, the cell suspension is provided in a syringe or a plurality of syringes.

In yet a further aspect the cell suspension is provided as a single dose.

In one embodiment of the cell suspension for the use according to present invention the treatment provides for a long-term improvement of axonal integrity in the white matter tracts of the brain of the subject after the stroke. In a preferred embodiment the treatment provides for a long-term improvement of the main brain tracts, such as the corticospinal tract or the corpus callosum.

In one embodiment of the cell suspension for the use according to present invention the treatment increases the neuro-restoration capacity of the brain of the subject. In one preferred embodiment the neuro-restoration capacity at the ischemic area of the brain is increased.

In a further preferred embodiment, the neuro-restoration capacity in the main white matter tracts located remotely from the area of the ischemic lesion is increased.

The present invention furthermore relates to a manufacturing process of a cell suspension comprising the steps of: a. Collecting bone marrow (BM) from a human subject; b. Mixing the BM with an anticoagulant in a ratio of 1:3 to 1:6 of BM to anticoagulant, preferably wherein the anticoagulant is Anticoagulant Citrate Dextrose Solution (ACD- A); c. Removing plasma, red blood cells and granulocytes from the suspension obtained in b) via density gradient centrifugation; d. Washing the suspension of BM obtained in c) by adding a wash solution, said wash solution preferably comprising saline and human albumin in a 1:1 ratio, and removing the wash solution via density gradient centrifugation; e. Subjecting the solution obtained in step d) to a filtration step, preferably through a 50 pm filter, into a sterile container; f. Centrifugation of the cell solution obtained in step e) and resuspension of the cell pellet in lactated Ringer solution, preferably supplemented with 2.5% glucose (v/v) and 1% albumin; and g. Optionally, packing the cell solution obtained in step f) in a sterile syringe.

In a preferred embodiment the BM is mixed with an anticoagulant in a ratio of from 1:4 to 1:5.5 of BM to anticoagulant, preferably in a ratio of 1:5.

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS

As used herein "autologous" is understood as referring to a cell preparation where the donor and the recipient are the same individual. As used herein "adult bone marrow derived cells" is understood as a preparation comprising cells, which are not embryonic and are derived from bone marrow obtained from a human donor.

As used herein "cell suspension" is understood as a preparation of cells suspended in a liquid medium.

As used herein "cell suspension of adult bone marrow derived cells" is understood as a preparation of cells, which are not embryonic and are derived from bone marrow obtained from a human donor suspended in a liquid medium.

As used herein "Hematopoietic stem cells that express CD34" is understood as hematopoietic stem cells which express the surface marker CD34 and are identified as CD34 positive by CD34 antibody staining and flow cytometry.

As used herein "Cells that express CD31", or "cells that are CD31+ cells" are understood as cells that express the marker CD31 and are identified as CD31 positive (CD31+) by CD31 antibody staining and flow cytometry. CD31 is expressed on the surface of tumor cells and mediates the adhesion to endothelial cells.

As used herein "progenitor cells that express CD133", or "cells that are CD133+ cells" are understood as cells that express the marker CD133 and are identified as CD133 positive (CD133+) by CD133 antibody staining and flow cytometry.

As used herein "Cells that express CD146", or "cells that are CD146+ cells" are understood as cells that express the marker CD146 and are identified as CD146 positive (CD146+) by CD146 antibody staining and flow cytometry. CD146 is expressed on the surface of tumor cells and mediates the adhesion to endothelial cells.

As used herein "cells that are CXCR4+ cells" are understood as cells which express the surface marker CXCR4 and are identified as CXCR4 positive by CXCR4 antibody staining and flow cytometry. CXCR4 is a receptor for migration factor SDF-1. As used herein "cells that are VEGFR2+ cells" are understood as cells which express the surface marker VEGFR2 and are identified as VEGFR2 positive by VEGFR2 antibody staining and flow cytometry. VEGFR2 is a receptor of VEGF, which is an angiogenesis and vasculogenesis factor.

As used herein, "circulating endothelial cells (CECs)" are mature cells that are shed from blood vessels. The "circulating endothelial cells (CECs)" that express CD133-/CD31+/CD146+" are understood as CECs that do not express the surface marker CD133 but do express the surface markers CD31 and CD146 and are identified as CD31 and CD146 positive by antibody staining and flow cytometry but are identified negative for the surface marker CD133 by CD133 antibody staining and flow cytometry.

As used herein "mononuclear cells" is understood as any blood or bone marrow white blood cell (also referred to as leukocytes) having a round nucleus, thereby excluding granulocytes.

As used herein "ischemic stroke" is understood as the medical condition that happens when the blood supply to part of the brain is cut off. "Acute ischemic stroke" is understood as those stroke patients within the first week from stroke onset. "MCA stroke" is understood as those strokes that involves the middle cerebral artery (MCA) but could not only be restricted to the MCA territory, as some patients may have ischemic lesions in other regions of the brain.

As used herein "hemorrhagic stroke" is understood as the medical condition that happens when blood from an artery suddenly begins bleeding into the brain.

The term "about" in reference to a numeric value means +/- 20% of that numeric value. The term "about" in reference to a numeric value also includes +/- 10% of that numeric value. The term "about" in reference to a numeric value also includes +/- 5% of that numeric value. The term "about" in reference to a numeric value also includes +/- 1% of that numeric value.

The terms "comprise" and "comprising" are used in the inclusive, open sense, meaning that additional elements may be included. The term "comprises" also encompasses and may be used interchangeably with the terms "consists of" and "consists essentially of". As used herein the term "adult" it is meant that the stem cells are not embryonic. In one embodiment, "adult" means post-embryonic or "post-natal". With respect to the stem cells of the present invention, the term "adult stem cell" means that the stem cell is isolated from a tissue or organ of an animal at a stage of growth later than the embryonic stage. Adult stem cells are unlike embryonic stem cells, which are defined by their origin, the inner cell mass of the blastocyst. Adult stem cells according to the invention may be isolated from any non-embryonic tissue, and will include neonates, juveniles, adolescents and adult subjects. Generally, the stem cell of the present invention will be isolated from a non-neonate mammal, and for example from a non-neonate human. Preferably, the stem cells of the present invention are isolated from a human.

The term "isolated" indicates that the cell or cell population to which it refers is not within its natural environment. The cell or cell population has been substantially separated from surrounding tissue.

The marker profile of the new cell suspension product referred to in the present invention can be further defined by the presence and/or absence of additional markers, or by a specific profile of a combination of present and absent markers. In each case, the specific combination of markers may be present as a particular profile within a population of cells and/or a particular profile of markers on individual cells within the population.

The term "marker" as used herein encompasses any biological molecule whose presence, concentration, activity, or phosphorylation state may be detected and used to identify the phenotype of a cell.

Then, cells of the invention are positive for certain phenotypic markers and negative for others. By "positive", it is meant that a marker is expressed within a cell. In order to be considered as being expressed, a marker must be present at a detectable level. By "detectable level" is meant that the marker can be detected using one of the standard laboratory methodologies such as PCR, blotting or flow cytometry analysis as described.

The term "expressed" is used to describe the presence of a marker on the surface of or within a cell. In orderto be considered as being expressed, a marker must be present at a detectable level. By "detectable level" is meant that the marker can be detected using one of the standard laboratory methodologies such as PCR, blotting, immunofluorescence, ELISA or FACS analysis. "Expressed" may refer to, but is not limited to, the detectable presence of a protein, phosphorylation state of a protein or an mRNA encoding a protein. A gene is considered to be expressed by a cell of the invention or a cell of the population of the invention if expression can be reasonably detected after 30 PCR cycles, preferably after 37 PCR cycles, which corresponds to an expression level in the cell of at least about 100 copies per cell. The terms "express" and "expression" have corresponding meanings. At an expression level below this threshold, a marker is considered not to be expressed.

DETAILED DESCRIPTION

The problem addressed by the present invention is to provide patients that have suffered an acute ischemic stroke with an improved therapeutic alternative. Present invention therefore in one aspect relates to a cell suspension of autologous adult bone marrow derived cells as further defined by the aspects and preferred embodiments referred to below and its use for the treatment of such patients.

THE CELL SUSPENSION

The cell suspension according to the present invention is suitable for the treatment or amelioration of a subject that has suffered an ischemic or hemorrhagic stroke. More specifically, it has been surprisingly found that the cell suspension according to the present invention when used in the treatment or amelioration of a subject that has suffered from an ischemic stroke leads to many clinically relevant improvements. An amelioration in neurological deficit could be measured using the NIHSS (National Institute of Health Stroke Scale) and an improvement in daily activities could be shown using the Barthel index. Efficacy of the treatment was also shown by measuring an improvement of disability of the subject treated in the long term (i.e. after 3 and 6 months of the event) using the modified Rankin scale. Furthermore, high safety of the treatment could be shown, as no serious adverse events were observed. The inventors could show that the above advantages were obtained even with a single administration of the cell suspension of present invention as illustrated in more detail in the examples section. Furthermore, the observed biological effects provide for treatment of subjects having suffered from a hemorrhagic stroke. A hemorrhagic stroke causes an increase of neuronal death and a proinflammatory response. The treatment with the cell suspension of present invention causes an increase of proliferation of endogenous neural stem cells and angiogenesis leading to an improved recovery of the subject after an ischemic and also an hemorrhagic stroke. Furthermore, all patients underwent MRI studies at baseline and during the 6-month follow-up visits. The inventors could demonstrate for the first time that after treatment of present invention there is a significant improvement of axonal integrity of the different white matter tracts of the brain in the longterm. This fact reflects the neuro-restoration capacity of the treatment in the brain, not only at the ischemic area but also in the main white matter tracts located remotely from the ischemic lesion.

The present invention therefore in one aspect relates to a cell suspension comprising from 1 x 10⁷ to 8 x 10⁹ of autologous mononuclear cells derived from the bone marrow of a human subject, wherein of the total number mononuclear cells in said cell suspension i. 1.06% to 20.27% are hematopoietic stem cells that express CD34; ii. 5.64% to 30.63% express CD31; iii. 0.3% to 1.81% are progenitor cells that express CD133; iv. 0.42% to 9.95% express CD146; v. 2.71% to 71.68% are CXCR4+ cells and 0% to 2.19% are VEFGR2+ cells vi. 0% to 1,14% are CD133-/CD31+/CD146+ circulating endothelial cells (CECs) for use in the treatment or amelioration of a subject suffering or having suffered from an ischemic stroke or a hemorrhagic stroke.

In certain embodiments of present invention, the total number of mononuclear cells in the cell suspension may be from about 2 x 10⁷to about 4 x 10⁹, from about 5 x 10⁷to about 2 x 10⁹, from about 8 x 10⁷ to about 1 x 10⁹, from about 9 x 10⁷ to about 9 x 10⁸, from about 1 x 10⁸ to about 8.1 x 10⁸ mononuclear cells.

In yet further embodiments the cell suspension of present invention may contain a total number of mononuclear cells of from about 3 x 10⁷ to about 3 x 10⁹, of from about 4 x 10⁷ to about 2.5 x 10⁹, of from about 5 x 10⁷ to about 2 x 10⁹, of from about 6 x 10⁷ to about 1.5 x 10⁹, of from about 7 x 10⁷ to about 1.2 x 10⁹, of from about 8 x 10⁷ to about 1 x 10⁹, of from about 9 x 10⁷ to about 9.5 x 10⁸, of from about 9.5 x 10⁷ to about 9 x 10⁸, of from about 1 x 10⁸ to about 8.5 x 10⁸, of from about 1 x 10⁸ to about 8.1 x 10⁸. Methods of measurement of the cell suspension of present invention are well known in the prior art. Whilst the inventors have used the method as described in more detail in Example 1, other suitable method know to the skilled person may be equally used.

Table 1 below summarizes the phenotypic analysis of the cell suspension as provided to for each patient including an analysis of the following cell types:

CD34+ Hematopoietic stem cells

CD34+CD38- Early, non-committed HSCs

CD133+ Endothelial progenitor containing cell population

CD90+ Early hematopoietic stem cells

CD105+ Cells expressing a membrane glycoprotein known as endoglin

CXCR4+ Cells expressing the SDF-1 receptor

VEGFR2+ Cells expressing vascular endothelial growth factor receptor 2

CD31+CD146+CD133- Mature endothelial cells

CD34+VEGFR2+CD133+ Late outgrowth endothelial progenitor cells

CD34-CD105+CD90+CD73+ MSCs

Table 1: Phenotypic analysis of CMMo-lctus-2013 drug substance. Data collected in the Phase II

(study CMMo-lctus-2013) for 36 subjects.

Methods used: PHE = phenotypic analysis of drug substance (MACS Quant Analyzer 10, Miltenyi Biotec);

Abbreviations: CD34+: hematopoietic stem cells expressing CD34;

CD34+/CD38-: proportion of hematopoietic stem cells expressing CD34 that do not express

CD38;

CD45+/CXCR4+: proportion of leukocytes that express CD45 and also express CXCR4;

CD34+/CXCR4: proportion of hematopoietic stem cells expressing CD34 that also express CXCR4;

CD45+ /VEGF+: proportion white blood cells expressing VEGF; N/A = not available. In one preferred embodiment the cells of the cell suspension of the invention are suspended in a volume of from about 5 ml to 30 ml of a heparinized saline solution ora lactated Ringer solution, said solutions preferably comprising additionally about 1% albumin and/or about 2.5% glucose.

In one preferred embodiment the cells of the cell suspension of the invention are suspended in a volume of from about 5 ml to 30 ml of a lactated Ringer solution, said solution further comprising about 1% albumin and about 2.5% glucose.

In a further aspect the invention also provides a pharmaceutical composition comprising the cell suspension of the invention as defined above and optionally a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, and/or dispersion media. The pharmaceutically acceptable carrier can comprise a medium which supports cell viability and functionality. Such medium can be serum-free to avoid provoking an immune response in the recipient. In one embodiment the carrier can be buffered and pyrogen free. In a preferred embodiment the final pharmaceutical composition is sterile to ensure safe application to the subject.

In one preferred embodiment the pharmaceutical composition comprising the cell suspension as described herein above comprises further excipients. A particularly preferred excipients is lactated Ringer solution, preferably with the addition of glucose and albumin. Most preferred is a lactated Ringer solution with about 1% albumin and about 2.5% glucose. In a further preferred embodiment, the pharmaceutical composition comprises ACD-A (Anticoagulant Citrate Dextrose Solution) solution as an anticoagulant in a ratio of 1:3 to 1:6, more preferred in a ratio of 1:4 to 1:5.5, most preferred in a ratio of 1:5 of the BM volume.

In yet a further preferred embodiment the cell suspension has a sufficiently low viscosity to allow use of a syringe for its administration. In yet a further preferred embodiment the pharmaceutical composition comprising the cell suspension has a sufficiently low viscosity to allow use of a syringe for its administration.

In a further aspect of present invention, the cell suspension is provided in a syringe or a plurality of syringes. In a preferred embodiment the syringe or the plurality of syringes is/are pre-filled. In yet a further aspect the cell suspension is provided as a single dose. As could be shown the advantages arising from the administration of the cell suspension of present invention were already observed after a single administration, see for more detail in the examples section.

MANUFACTURING PROCESS OF THE CELL SUSPENSION

The present invention furthermore relates to a manufacturing process of a cell suspension comprising the steps of: a. Collecting bone marrow (BM) from a human subject; b. Mixing the BM with an anticoagulant in a ratio of 1:3 to 1:6 of BM to anticoagulant, preferably wherein the anticoagulant is Anticoagulant Citrate Dextrose Solution (ACD- A); c. Removing plasma, red blood cells and granulocytes from the suspension obtained in b) via density gradient centrifugation; d. Washing the suspension of BM obtained in c) by adding a wash solution, said wash solution preferably comprising saline and human albumin in a 1:1 ratio, and removing the wash solution via density gradient centrifugation; e. Subjecting the solution obtained in step d) to a filtration step, preferably through a 50 pm filter, into a sterile container; f. Centrifugation of the cell solution obtained in step e) and resuspension of the cell pellet in lactated Ringer solution, preferably supplemented with 2.5% glucose and 1% albumin; and g. Optionally, packing the cell solution obtained in step f) in a sterile syringe.

In a preferred embodiment of the method of the present invention the BM is mixed with an anticoagulant in a ratio of 1:4 to 1:5.5, most preferred in a ratio of 1:5 of BM to anticoagulant.

The cell suspension of the invention can be manufactured as follows:

Bone marrow harvesting was performed obtaining 100 milliliters or 150-350 milliliters in the low- dose and high-dose groups, respectively using an Acid Citrate Dextrose solution A (ACD-A) at a proportion of 1:5. Cell production was performed following Good Manufacturing Practices (GMP) using an automated device SEPAX to ensure quality controlled cell production and to minimize risks. The harvest bone marrow was centrifuged on a Ficoll density gradient to isolate the mononuclear cells and obtain the low and high doses (2x106 per kilogram or 5x106 per kilogram), which were washed and resuspended in 20 ml of lactated Ringer solution supplemented with 1% heparin and 2,5% of glucose and packaged in two sterile syringes, with 10 ml each, for infusion into the patient.

Manufacturing is a continuous process and does not involve any activation steps or other extensive manipulations of cells that may affect functionality of the specific cell populations within the MNC fraction. Process duration may affect functionality of cells and therefore, preferably no hold times are incorporated in the process and the entire process is preferably completed within 4-5 hours.

APPLICABILITY OF THE CELL SUSPENSION

Results as shown in example 3 show that within the first 6 months following the administration of the cell suspension of present invention the treatment as described herein above leads to many clinically relevant improvements, such as amelioration in neurological deficit, an improvement in daily activities and a long-term improvement of disability of the subject treated. Furthermore, high safety of the treatment could be shown. Importantly, the inventors could show that the above advantages were obtained even with a single administration of the cell suspension of present invention.

Hence, a further aspect of the invention refers to the cell suspension, the pre-filled syringe or the pharmaceutical composition as defined in the section entitled "THE CELL SUSPENSION PRODUCT" for use in the treatment or amelioration of stroke patients, preferably ischemic or hemorrhagic stroke patients, most preferred acute stroke patients.

Furthermore, in one aspect of the present invention the cell suspension or the pharmaceutical composition as described herein above is administered into the stroke related area via intraarterial administration. The intra-arterial route is preferred as it is known to increase the direct contact with the endothelial cells at the ischemic tissue and thus leads to a wider distribution along the ischemic lesion in the brain when compared to the intravenous route. In one preferred embodiment the cell suspension or the pharmaceutical composition is administered intra-arterially at a rate of between 0.2 ml - 1.5 ml/min, preferably of between 0.5 ml-1 ml/min into the stroke related area.

In one aspect of the embodiments of present invention the cell suspension or the pharmaceutical composition is administered into the stroke related area via intra-arterial administration. In a preferred embodiment the cell suspension is administered into the middle cerebral artery (MCA), the anterior cerebral artery (ACA), or the posterior cerebral artery (PCA).

The inventors could also demonstrate that after the treatment with the cell suspension of present invention there is a significant long-term improvement of the different tracts of the brain showing that the treatment increases the neuro-restoration capacity of the brain not only at the ischemic area but also in the main white matter tracts located remotely from the ischemic lesion. In one embodiment of the cell suspension for the use according to present invention the treatment provides for a long-term improvement of axonal integrity of the white matter tracts of the brain of the subject after stroke. In a preferred embodiment the treatment provides for a long-term improvement of the main brain tracts, such as the corticospinal tract or the corpus callosum.

In one embodiment of the cell suspension for the use according to present invention the treatment increases the neuro-restoration capacity of the brain of the subject.

In one preferred embodiment the neuro-restoration capacity at the ischemic area of the brain is increased.

In a further preferred embodiment, the neuro-restoration capacity in the main white matter tracts located remotely from the area of the ischemic lesion is increased. EXAMPLES

Example 1: Method of producing the cell suspension from Bone Marrow and composition of Cell suspension

The procedure for obtaining the autologous bone marrow was carried out in a room equipped with positive pressure or in an operating room to guarantee maximum sterile conditions.

The patient's bone marrow was obtained by repeated aspirations over the posterior iliac crest under local anesthesia plus sedation until a volume of approximately 100 ml to 200 ml of marrow was obtained, depending on the assigned treatment group. In cases where, in the opinion of the physician, this volume was considered insufficient to manufacture the corresponding dose, a larger volume was withdrawn, up to a maximum of 350 ml. The obtained marrow was collected in a transfer bag containing ACD-A (Anticoagulant Citrate Dextrose Solution) solution as an anticoagulant in a ratio of 1:5 of the BM volume. The further processing of the BM consists only of the removal of plasma, red blood cells and granulocytes, obtaining only mononuclear cells (BM-MNCs). The procedure was carried out by means of density gradient centrifugation using Ficoll-Hypaque of density 1077 in a SEPAX automated cell processing system. The obtained BM- MNC suspension was subjected to two washes with a wash solution composed of 250 ml saline + 250 ml human albumin in the same machine in order to remove Ficoll. At the end of this process, approximately 50 ml of cell suspension of MNCs were obtained. The latter cell suspension was filtered through a sterile 50-micron plastic filter to avoid microaggregates. The MNCs obtained were transferred to a sterile 50 ml plastic tube and centrifuged. The cell pellet was resuspended in the same tube with packing medium consisting of lactate Ringer's solution supplemented with 2.5% glucose and 1% albumin.

Cells resuspended in packaging medium are packed in two sterile syringes of 10 ml each (primary packaging). The syringes were properly sealed with sterile plastic luer-lock caps that are individually labelled and placed in sterile rotation-seal plastic bags (secondary packaging) that are appropriately labelled. Under normal conditions the cell product was immediately infused into the patient. If for any reason it was necessary to store the product for a certain period of time, this was done at 2-8^C in a refrigerator with a permanent graphic temperature register and the time of entry and exit of the drug from this equipment was recorded. Throughout the procedure, which normally has a total duration of approximately three to four hours, aliquots of the cell product were extracted for the following controls:

• Sample of the initial product, i.e., pre-processed bone marrow, for haemocytometry and blood culture studies.

• Sample of the fractionated and washed product before final volume reduction centrifugation for haemocytometry and blood culture studies, as well as determination of cell viability by Trypan Blue.

• Sample of the supernatant of the final product for Gram stain.

All handling will be carried out under strict sterile conditions in a laminar flow cabinet located in the processing laboratory with HEPA filtered air.

To illustrate the present invention, a cell suspension according to the present invention has been used in the following examples made of an autologous cell suspension of BM-MNCs composed of several mature cell types as well as hematopoietic progenitor cells. The formulation of the final product was based on the number of viable WBCs present.

Characterization of the cell suspension of the invention was conducted with aliquots of BMMNCs (1-5 x 105 cells) that were stained with fluorochrome-conjugated monoclonal antibodies against the human cell surface markers CD34-FITC (fluorescein) and CD133-APC (allophycocyanin) (Miltenyi Biotec). After incubation for 15 minutes in the dark, the BMMNCs were washed with PBS and centrifuged for 5 minutes at 360 g. The cell pellet was then resuspended in phosphate buffered saline (PBS) and stained with propidium iodide (Miltenyi Biotec) to measure cell viability. A MACSQuant cytometer with MACSQuantify software was used to analyze the results for quantitative measurements.

The concentration of mononuclear cells expressing different markers from 36 clinical batches are presented in Table 1.

EXAMPLE 2: Study design and patients

The IBIS trial is a multicenter prospective phase lib, randomized, controlled (non-treated group as control), assessor-blinded, academic clinical trial of intra-arterial transplantation of autologous BM-MNC in acute ischemic stroke patients (PROBE design). A detailed description of the study design is published elsewhere¹¹. The study flow chart is shown in figure 1. Selection of study population

Between April 2015, and May 2021, 77 patients with ischemic stroke in the middle cerebral artery territory were randomized. Criteria for inclusion were age between 18 and 80 years, National Institute of Health stroke scale (NIHSS) score of 8-20 at inclusion, and treatment window within 7 days of stroke onset. In every patient, ipsilateral carotid artery and MCA permeability was proven before inclusion. Diffusion-weighted MRI (DWI) confirmed then non-lacunar ischemic nature of the MCA stroke in every case.

Patients with lacunar or hemorrhagic stroke were excluded. Other exclusion criteria were pregnancy, childbearing potential, history of present or previous malignant disease during the last 5 years, life threatening illness, stroke of haematological cause, significant previous disability (pre-stroke mRankin Scale score >3), and severe co-morbidity that would prevent follow-up.

This study was done in accordance with the Declaration of Helsinki and the Good Clinical Practice Guidelines and approved by the National Ethics Committee and the National Regulatory Agency (EudraCT : 2013-002135-15). Written informed consent was obtained from each patient or their representatives.

Randomization and masking

After written inform consent, patients were randomized 2:1:1 to control group (standard stroke treatment) or intra-arterial BM-MNC transplantation with low BM-MNC dose (2x106 per kilogram) or high dose (5x106 per kilogram) based on a central computer-generated code list. Patients were stratified at randomization by severity of neurological deficit measure by NIHSS (NIHSS score of 6-13 and 14-20) and center of cell production (Hospital Universitario Virgen del Rocio and Hospital Universitario Reina Sofia). The allocation was unblinded to patients and investigators. At each center, neurologists assessing patients during follow-up were unaware of the treatment allocation.

Treatment Procedures

Transplantation procedure was done within 1 to 7 days from stroke onset as previously described¹⁰. In brief, once the patient was in the operating room and under sedation and analgesia, bone marrow harvesting was performed obtaining 100 milliliters or 150-350 milliliters in the low-dose and high-dose groups, respectively using an Acid Citrate Dextrose solution A (ACD-A) at a proportion of 1:5. Cell production was performed following Good Manufacturing Practices (GMP) using an automated device SEPAX to ensure quality controlled cell production and to minimize risks. The harvest bone marrow was centrifuged on a Ficoll density gradient to isolate the mononuclear cells and obtain the low and high doses (2xl0⁶ per kilogram or 5xl0⁶ per kilogram), which were washed and resuspended in 20 ml of lactated Ringer solution supplemented with 1% heparin and 2,5% of glucose and packaged in two sterile syringes, with 10ml each, for infusion into the patient. Aliquots of the final BM-MNC product was separated for cell counting, flow cytometry and bacterial culture.

Within 24 hours after bone-marrow harvest, a cerebral angiography (3000 U heparin) was done and BM-MNCs were infused through microcatheter in the Ml segment of the infarct-related MCA at a rate of 0.5-lmL/min. The final suspension of BM-MNC was shaken until moment of infusion to avoid precipitating or clumping. After injection, a control angiography was done to ensure MCA patency.

No bone marrow aspiration, angiography, or sham injection was performed in the control group. Every patient received medication according to current guidelines.

At baseline screening, demographic and medical history, acute stroke treatments (thrombolysis and thrombectomy), vital signs, clinical scores (pre-stroke mRS, NIHSS), and laboratory data were recorded. In the experimental group, during bone marrow harvest and intra-arterial infusion, vital signs and neurological status were monitored.

Clinical and functional evaluation (mRankin Scale, Barthel index and NIHSS), blood analysis, and recording of adverse events were performed the day after transplantation (or randomization in the control group), at hospital discharge, and 1, 3, and 6 months after the stroke. A follow-up MRI was performed at 6 months in both groups.

Outcomes

The prespecified primary outcome was the proportion of patients with modified Rankin Scale (mRS) scores of 0-2 at 180 days in the intention-to-treat population. Pre-specified secondary outcomes were categorical shift in mRS ordinal (0-6) scale, mRS scores of 0-3 at 6 months, and NIHSS and Barthel scores at 1-3-6 months. As safety endpoint, proportion of adverse events and serious adverse events in each group was analyzed.

Statistical analysis

Sample size was calculated based on a previous trial (8). The total sample size of 76 patients, with 38 patients in each arm, provides an 80% power to detect a difference of 18% in dependency in mRS. The Kolmogorov-Smirnov test was applied to verify if the variables followed a normal distribution. When the variables were found not to be normally distributed, comparisons between groups of data were made using Mann-Whitney U tests to detect differences in the distribution of samples, and Spearman's Rho coefficient to assess the relationship between two quantitative variables. Categorical data are expressed as percentages and analyzed using the Chi- square test (x2) or Fisher's exact test where appropriate. Categorical shift in mRS was undertaken on the full range (0-6) of the mRS using Cochran-Mantel-Haenszel shift test and proportional odds logistic regression subject to the validity of shift analysis model assumptions.

Primary and secondary efficacy analysis was done in the intention to treat population, which included all patients who underwent randomization and were not lost to follow-up. Safety analysis was performed in the per protocol population. We repeated efficacy analysis with per protocol population. All statistical analyses were performed using the SPSS software package version 15.0 for Windows (SPSS Inc., Chicago, IL, USA). Differences were considered to be statistically significant when two-tailed P values were less than 0.05.

The trial is registered with ClinicalTrials.gov, number NCT 02178657. The trial was approved by the Spanish Agency of Medicines and Medical Devices (AEMPS)(EudraCT:2013-002135-15).

Example 3: Results and Discussion

Patients with ischemic stroke in the middle cerebral artery territory were assessed for eligibility (figure 1). Of them, 37 were excluded and 77 patients were randomized. Baseline characteristics and stroke severity were similar in both groups, with a mean age of 62-4, and 59.7% were men, see table 2 below.

Table 2: Baseline characteristics: Data are number (%) or mean (SD). NIHSS=National Institutes of Health stroke scale. MCA=Middle cerebral artery. TOAST=TOAST (Trial of Org 10172 in Acute Stroke Treatment) classification.

Most of patients received recanalization therapy at the hyperacute phase of stroke, with 45.5% treated with intravenous thrombolysis and 81.8% with mechanical thrombectomy (86.8% in control group vs 80% in low-dose group and 73.7% in high-dose group, p=0.46). However, the randomized patients still had disabling deficits with a median NIHSS at inclusion of 12 [9-15], p=0.94 (after a mean of 3.4 days from stroke onset). In baseline MRI, mean infarct volume was 75.4 ±39.1mL, with 55 patients (71.4%) with a left MCA stroke. There were no significant differences in stroke etiology (p=0.17). Time from stroke onset to inclusion in the trial was well balanced between group with a mean of 3.4 ±1.2 days (p=0.85). In the BM-MNCs patients, intra-arterial BM-MNCs injection was done at a mean of 5-7 days after stroke onset in the low-dose group and 5.5 days in the high-dose group (p=0.67). One patient randomized to the low-dose cell group, did not received the BM-MNCs intra-arterial injection after bone marrow harvest, due to technical problems with cell production. Another patient randomized to the high-dose cell group, received finally the low-dose of cells, as there were not enough cells to inject the 5xl0⁶ per kilogram dose.

Of the total BM-MNCs injected, a mean of 7.42 xl0⁶(±3.97) were CD34+ cells and 0.40xl0⁶(±0.49) were CD133+ cells. All cultures were negative for bacteria.

In the safety analysis, there were no differences in the rate of adverse events and serious adverse events, see table 3 below.

Table 3: Adverse events: Data are number (%)

No serious adverse events were related to the cell therapy. Two patients had minor femoral hematoma after intra-arterial injection of cells, with no other adverse events related to cell therapy.

In the intention-to-treat analysis, the primary outcome occurred in 38.9% in control group and 47.4% in the BM-MNC group (adjusted OR 2.22 [95% Cl 0.72-6.85], p=0.16), with 50.0% in the 2xl0⁶/kg dose group and 44.4% in the 5xl0⁶/kg dose group (aOR 2-08 [95% Cl 0-55-7-85], p=0-28 and aOR 1-89 [95% Cl 0-52-6-96], p=0-33, respectively). There were no significant differences between groups in mRS 0-3, in shift analysis, in NIHSS, or Barthel scores at 180 days, see Table 4 depicted in Figure 3.

In the per-protocol analysis, results in primary and secondary outcomes were similar to the intention-to-treat analysis, but there was a trend towards lower neurological deficits in the NIHSS and higher Barthel index scores at 6 months in the low-dose group (p=0.07 and p=0.08, respectively). In post-hoc analysis of efficacy at 90 days, the mRS of 0-2 was 32.4% in control group and 47.4% in BM-MNCs group (aOR 3.72 (1.06-13.02), p=0.04), with a rate of 55.0% in the low-dose group and 38.9% in the high-dose group (aOR 4.46 (1.04-19.10), p=0.04 and OR 2.97 (0.66-13.27), p=0.15). A trend towards higher odds of improvement of 1 point in the shift analysis of the mRS was found in the per-protocol analysis in the BM-MNCs group (p=0.09), especially in the 2xl0⁶/kg dose group p=0.07).

The infarct volume measure in the basal visit was similar in the three groups, with a mean volume of 75.5±40.9mL in the control group vs 73.7±35.7 in the 2xl0⁶/kg dose group (p=0.88) and 78.7±42.7 in the 5xl0⁶/kg dose group (p=0.80). In 50 patients we could perform the 6-months MRI follow-up and measure the volume change. There were no significant differences in infarct volume change in the three groups (29.4±18.7mL in control group vs 20.4±19.8 in the 2xl0⁶/kg dose group, p=0.16; and 20.3±11.2 in the 5xl0⁶/kg dose group, p=0.13).

In summary, the inventors herein describe the biggest clinical trial to date which evaluates the efficacy of cell therapy injected intra-arterially in stroke patients. In this trial they explored the effect of the administration of a specific BM-MNCs cell suspension as further detailed above using two different doses injected intra-arterially within the first days after an ischemic stroke.

They could show that within the first 6 months following the administration of the cell suspension of the invention as described herein above leads to significant and clinically relevant improvements, such as the amelioration in neurological deficit, an improvement in daily activities and a long-term improvement of disability of the subject treated. Furthermore, high safety of the treatment could be shown.

Specifically, the inventors observed better outcomes in mRS, Barthel, and NIHSS in both doses of BM-MNCs. A total difference of 9.7% in the rate of good outcomes in primary outcome (mRS 0-2 at 6 months) between BM-MNCs-treated patients and control group was detected. The results seem to be more pronounced in the low-dose group (2xl0⁶/kg BM-MNCs) with an absolute difference of 11.1% of improvement in long-term disability measured with mRS, and with a trend towards better Barthel index scores and lower neurological deficit in the NIHSS in the per- protocol analysis. Also, in the analysis of the mRS at 3 months a significant increase was showed in the rate of independent patients in the low-dose group (aOR 4.46, p=0.04). Contrary to preclinical data, the inventors could not see a direct dose-response relationship.

There were no significant safety issues during the trial, including the bone marrow harvest and cell infusion and the 6-months follow-up. No differences in the percentage of AEs or SAEs were detected between groups.

In conclusion, intra-arterial BM-MNCs in acute ischemic stroke patients was safe and well tolerated, in a population with high rate of recanalization therapies in the acute stroke phase.

Example 4: MRI sub-study

Setup of the study

All patients underwent MRI studies baseline and 6-month follow-up visits according to study protocol. According to the center feasibility, the studies were acquired on 3 different MRI machines; 33 patients were on a 1.5T (Intera Philips), 37 patients on a 1.5T (Aera Siemens) and 7 patients on 3T (Ingenia Philips). Subjects wore professional anti-noise earplugs to protect their hearing and were bilaterally fixed with sponge pads to reduce motion artifacts. Protocol included axial Tl-weighted image, axial T2-weighted image, axial T2 gradient-echo weighted image, three- dimensional fluid-attenuated (FLAIR), and DTI images. On 1.5T MRI studies, FLAIR images were acquired with TR= 4800ms, TE= 1660ms, inversion time= 350ms, slice thickness = 1.2mm. On 3.0T studies, setting was TR= 4800ms, TE= 1650ms, inversion time= 302.69ms, slice thickness = 1.12mm.

DTI was acquired using a single-shot spin echo sequence with echo-planar imaging (EPI), 60 contiguous slices, voxel size 1.5x1.5x2 mm3, TE/TR of 75/4500 ms/ms, a diffusion-weighting factor b = 800 s/mm2 and diffusion encoding along 16 directions.

Material and Methods a) Image processing

The segmentation of volumes of interest (VOI) of stroke lesions was performed using the software Imfusion Labels (ImFusion GmbH, Munich, Germany). Using the blOOO of diffusion weighted imaging sequence for the baseline volumes and FLAIR for the follow up 6 months. The segmentations were done semi-automatic and revised by a radiologist with more of 20 years of expertise. The DTI parametric maps were calculated using the software DSI Studio. After that quality check was performed on each individual space to ensure the correct values. 17 patients were excluded from study by artefacts, errors on reconstruction or missing data. These maps were co-registered and normalized to the MNI standard space with template of each of these maps by SPM12.

Lesions segmentations mask were subtracted from the DTI parametric maps to get the mean values of each tract without effect of the lesion. DTI-metrics, such as fractional anisotropy (FA), axial diffusivity, radial diffusivity and mean diffusivity, were extracted for each tract from the ICBM DTI-81 atlas using the brain software library (FSL). The results are shown in Figures 5 and 6. b) Outcome

The primary outcome was the variation from baseline to follow-up in FA. The secondary outcome was the differential variation in stroke volume by treatment status.

Initial comparisons were performed between the control group and the two both experimentally treated groups. Subsequently, a comparison was made between the experimental and control patients. c) Statistical Analysis

Normality testing was performed using the Shapiro-Wilk test. Statistical significance was assumed at P<0.05. All data were analyzed using SPSS version 24.0 (SPSS, Inc, Chicago, IL) and MATLAB R2016a (Mathworks, Natick, MA).

Results of the study

In the baseline and 6-month studies, no statistically significant differences in infarct volumes were detected. At six months, however, patients treated with BM-MNCs experienced a greater reduction in infarct volume in the control study than in the baseline study.

In baseline, DTI study evaluated by fractional anisotropy demonstrated non-statistical differences in baseline characteristics of the tracts (table 6) between the control group versus de BM-MNCs group. When the variations in fractional anisotropy of the tracts from baseline to 6 months of follow-up were analyzed, most of the tract analyzed improved in long-term (i.e., 6-months followup) in the cell therapy group compared with the control group (table 7).

Conclusion

The inventors could demonstrate for the first time in the setting of a randomized clinical trial with cell therapy in stroke, that there is a significant improvement of axonal integrity of the different white matter tracts of the brain in the long-term. This fact reflects the neuro-restoration capacity of the treatment in the brain, not only at the ischemic area but also in the main white matter tracts located remotely from the ischemic lesion. REFERENCES

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Claims

1. A cell suspension comprising from about 1 x 10⁷ to about 8 x 10⁹ of autologous mononuclear cells derived from the bone marrow of a human subject, wherein of the total number of mononuclear cells in said cell suspension i. 1.06% to 20.27% are hematopoietic stem cells that express CD34; ii. 5.64% to 30.63% express CD31; iii. 0.3% to 1.81% are progenitor cells that express CD133; iv. 0.42% to 9.95% express CD146; v. 2.71% to 71.68% are CXCR4+ cells and 0% to 2.19% are VEFGR2+ cells vi. 0% to 1,14% are CD133-/CD31+/CD146+ circulating endothelial cells (CECs) for use in the treatment or amelioration of a subject suffering or having suffered from an ischemic stroke or a hemorrhagic stroke.

2. The cell suspension for use according to claim 1, wherein the cell suspension comprises about 2 x 10⁷to about 4 x 10⁹ mononuclear cells, preferably about 5 x 10⁷to about 2 x 10⁹ mononuclear cells.

3. The cell suspension for use according to claim 1, wherein the cell suspension comprises about 8 x 10⁷to about 1 x 10⁹ mononuclear cells, preferably about 9 x 10⁷to about 9 x 10⁸ mononuclear cells.

4. The cell suspension for use according to claim 1, wherein the cell suspension comprises about 1 x 10⁸ to about 8.1 x 10⁸ mononuclear cells.

5. The cell suspension for use according to any one of claims 1 to 4, the cell suspension further comprising a lactated Ringer solution, said lactated Ringer solution preferably comprising about 1% albumin and/or about 2.5% glucose.

6. The cell suspension for use according to any one of claims 1 to 5, wherein the ischemic stroke is selected from a middle cerebral artery (MCA) ischemic stroke or ischemic lesions in other regions of the brain, preferably the anterior cerebral artery (ACA) or the posterior cerebral artery (PCA).

7. The cell suspension for use according to any one of claims 1 to 6, wherein the ischemic stroke is an acute ischemic stroke.

8. The cell suspension for use according to any one of claims 1 to 5, wherein the hemorrhagic stroke is selected from lobar hemorrhagic stroke or deep hemorrhagic stroke, preferably wherein the hemorrhagic stroke is an acute hemorrhagic stroke.

9. The cell suspension for use according to any of claims 1 to 8, wherein the cell suspension is administered into the stroke related area via intra-arterial administration.

10. The cell suspension for use according to claim 9, wherein the intra-arterial administration is performed at a rate of between 0.2 ml - 1.5 ml/min, preferably of between 0.5 ml - 1 ml/min into the stroke related area.

11. The cell suspension for use according to any of claims 9 or 10, wherein the intra-arterial administration is performed between 1 to 7 days after the onset of the stroke.

12. The cell suspension for use according to any one of claims 1-11, wherein the cell suspension is provided in a syringe or a plurality of syringes.

13. The cell suspension for use according to any one of claims 1-12, wherein said cell suspension is provided as a single dose.

14. A manufacturing process of a cell suspension comprising the steps of: a. Collecting bone marrow (BM) from a human subject; b. Mixing the BM with an anticoagulant in a ratio of between 1:3 to 1:6 of BM to anticoagulant, preferably wherein the anticoagulant is Anticoagulant Citrate Dextrose Solution (ACD-A); c. Removing plasma, red blood cells and granulocytes from the suspension obtained in b) via density gradient centrifugation; d. Washing the suspension of BM obtained in c) by adding a wash solution, said wash solution preferably comprising saline and human albumin in a 1:1 ratio, and removing the wash solution via density gradient centrifugation; e. Subjecting the solution obtained in step d) to a filtration step, preferably through a 50 pm filter, into a sterile container; f. Centrifugation of the cell solution obtained in step e) and resuspension of the cell pellet in lactated Ringer solution, preferably supplemented with 2.5% glucose and 1% albumin; and g. Optionally, packing the cell solution obtained in step f) in a sterile syringe.