METHOD OF PRODUCING MACROPHAGES

Abstract

The improved 4-5 day, optionally 3-5 day GMP-compliant in-vitro method enables the production of macrophages from monocytes that benefits from a shorter cell culture time, fewer interventions whilst maintaining the desired characteristics of the human macrophages. The present invention describes a method wherein the monocytes are cultured in medium comprising one or more growth actors to stimulate macrophages with a pro-regenerative phenotype. The method described herein is xeno-free, serum-free and GMP compliant. In addition, further disclosed are macrophages produced according to the present invention and the use of said macrophages in the treatment of liver diseases, such as liver cirrhosis.

Claims

1. An in vitro GMP-compliant method of producing macrophages comprising: (a) culturing monocytes in medium for 3-5 days to produce macrophages, wherein the medium comprises one or more growth factors to stimulate macrophage production; and wherein step (a) takes place entirely in the same medium.

2. The method according to claim 1, wherein the monocytes are cultured for 5 days.

3. The method according to any preceding claim, wherein the method does not comprise re-feeding or replacing medium.

4. The method according to any preceding claim wherein the monocytes are seeded at a density of between 1×10.sup.6 cells/cm.sup.2 up to 1×10.sup.7 cells/cm.sup.2.

5. The method according to any preceding claim, wherein the medium is selected from X-Vivo 10, X-Vivo15, TexMACS, AIMv, RPMI, DMEM, and DMEM/F12, preferably TexMACS.

6. The method according to any preceding claim wherein the medium comprises one or more factors selected from the CSF family, preferably M-CSF.

7. The method according to any preceding claim, wherein the medium contains M-CSF at a concentration of between 25-200 ng/mL.

8. The method according to any preceding claim wherein the monocytes are human, and the macrophages are human monocyte derived macrophages (hMDMs).

9. The method according to any preceding claim wherein the monocytes are derived from human blood, preferably the buffy coat of human blood, preferably from the PBMC fraction of human blood.

10. The method according to any preceding claim, wherein the method further comprises a step of polarisation of the macrophages produced in step (a), preferably into M1-like or M2-like macrophages.

11. The method according to claim 10, wherein the further step of polarisation of the macrophages comprises a step of polarising factors added to the medium, preferably M1 or M2 polarising factors.

12. The method according to claim 11, wherein the M1 polarizing factors are selected from: GM-CSF, IFNγ, and TLR agonists such as LPS; and the M2 polarizing factors are selected from: IL10, IL4, IL13, and poly(I:C).

13. The method according to any preceding claim, wherein the method produces mature macrophages with a yield of at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%,least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%.

14. A macrophage produced by the method according to any of claims 1-13.

15. An ex vivo generated macrophage having a pro-regenerative phenotype, optionally produced by the method according to any of claim 1-13.

16. The macrophage according to claim 14 or 15, wherein the macrophage has increased expression of one or more pro-regenerative cytokines.

17. The macrophage according to any of claims 14-16, wherein the macrophage has increased expression of one or more anti-inflammatory cytokines and/or reduced expression of one or more inflammatory cytokines, and/or increased expression of one or more anti-fibrogenic cytokines, and/or reduced expression of one or more fibrogenic cytokines.

18. The macrophage according to any of claims 14-17 wherein the macrophage has reduced expression of one or more of the following cytokines: IL1, IL12, IL17 (A, B, C, F), IL18, TNFα, IFNγ, preferably IL17F.

19. The macrophage according to any of claims 14-18, wherein the macrophage has reduced expression of one or more of the following cytokines: IL4, IL13, PDGF, TGFβ (1, 2, 3), preferably TGFβ1.

20. The macrophage according to any of claims 14-19 wherein the macrophage expresses mature cell surface markers, preferably CCR2−, CD14+, CD206+, CD163+, CD169+, 25F9+, and CD86+.

21. A population of macrophages according to any of claims 14-20.

22. A composition comprising a population of macrophages according to claim 21.

23. A macrophage, population or composition according to any of claims 14-22 for use as a medicament.

24. A macrophage, population, or composition according to any of claims 14-22 for use in the treatment of disease or injury, wherein the disease is selected from the list comprising liver disease, kidney disease, lung disease or muscle disease.

25. A macrophage, population, or composition for use according to claim 24, wherein the liver disease, kidney disease, lung disease or muscle disease is a fibrotic disease or an inflammatory disease, optionally the disease is acute or chronic, optionally selected from the list comprising a drug overdose preferably APAP overdose and liver cirrhosis.

26. A macrophage, population or composition according to any of claims 16-24 which respond to inflammatory stimuli such as one or more of: IFNγ, IL10, IL4, IL13, and LPS.

Description

FIGURES

[0260] FIG. 1: shows characterisation of the inventive protocol (day5 no feed) vs. standard protocol (day 7 plus re-feed) A Representative pictures of day5 no feed macrophages (day5) and day7 plus feed macrophages (day7) from two distinct donors (brightfield, 20×, scale bar=200 mm); B Yield of the day5 no feed vs. the day7 plus feed protocol. The percentage (%) yield is calculated as: (Number of macrophages at harvest/number of macrophages plated)*100. D'Agostino and Pearson omnibus normality test was carried out, and then a non-parametric Mann Whitney test was applied. p=0.0641; C Viability at the end of the day5 no feed vs. the day7 plus feed protocol. The percentage (%) is calculated as: % of DRAQ-CD45+ macrophages in the single cell gate. D'Agostino and Pearson omnibus normality test was carried out, and then a non-parametric Mann Whitney test was applied. p=0.99; D-E Gas chromatography results on supernatants of five day5 no feed and five day7 plus feed cell culture supernatants. Glucose (D) and lactate (E) are measured. Sample too small to carry out a D'Agostino and Pearson omnibus normality test: a non-parametric Mann Whitney test was applied. p=0.99; F Adhesion properties of the day5 no feed and day7 plus feed macrophages. The percentage (%) of adhesive cells is calculated as: (number of plated cells pre-incubation and washing/number of harvested cells post-incubation and washing)*100. Sample too small to carry out a D'Agostino and Pearson omnibus normality test: a non-parametric Wilcoxon signed rank test was applied. p=0.1875.

[0261] FIG. 2: shows further characterisation of the inventive protocol (day5 no feed) vs. standard protocol (day 7 plus re-feed) A Dosage of functionally relevant cytokines using the V-Plex technology. Data are expressed in pg/mL. Day5 and day7 concentrations are calculated as reported in materials and methods. The same donors are tested for day5 and day7 cytokine secretion. Data are analysed using t-test for paired data. **p<0.01; ***p<0.001; B Flow cytometry analysis of cell surface markers. Each dot represents a donor. MFI ratio is calculated dividing the day5 or day7 MFI by the MFI of CD14+ monocytes at the start of the culture. The dotted line represents a ratio of 5, considered the minimal target ratio for CD206 and 25F9 to release the day7 macrophages for treatment. Data are analysed using t-test for unpaired data; C Phagocytosis assay using live imaging. We report the mean±SD of the macrophages' cytoplasmic MFI in the AlexaFluor488 channel (i.e. of the ingested beads). There are no significant differences at any of the data point analysed. Data are analysed using a two-way ANOVA.

[0262] FIG. 3: shows further characterisation of the inventive protocol (day5 no feed) vs. standard protocol (day 7 plus re-feed) A Yield of day5 and day7 protocols with and without feed. Each symbol is an independent donor; B Yield of day5 and day7 protocols with and without feed, with and without human serum type AB (hserum AB); C Gas chromatography analysis of Glucose in the cell culture supernatants of hMDMs cultured in AimV (AV) or TexMacs (TM) with or without feed, with or without serum. Each symbol is an independent donor; D Gas chromatography analysis of Lactate in the cell culture supernatants of hMDMs cultured in AimV (AV) or TexMacs (TM) with or without feed, with or without serum. Each symbol is an independent donor; E Viability of cells using day5 protocols with and without feed, with and without human serum type AB (hserum AB) F Day-by-day analysis of viability by flow cytometry in two donors using a no-feed and a feed protocol. All data are reported as mean±SD.

[0263] FIG. 4: shows characterisation of the day-by-day differentiation of monocytes into macrophages, with or without feeding, A-H Flow cytometry analysis of cell surface markers. At each time point, each symbol represents monocyte-macrophages from a single donor over time. Monocyte-macrophages with and without feed are represented with different symbols (). MFI ratio is calculated dividing the day.sub.x MFI by the MFI of CD14+ monocytes at the start of the culture. The specific marker analysed is reported in the label of the Y axis.

[0264] FIG. 5: shows characterisation of the inventive protocol (day5 no feed) vs. standard protocol (day 7 plus re-feed) A Dosage of functionally relevant cytokines using the V-Plex technology. Data are expressed in pg/mL. Data from day5 and day7 polarised macrophages are clusterised using the online platform Morpheus. The same donors are tested for day5 and day7 cytokine secretion. The false-colour scale ranges from light grey to dark grey to represent minimal to maximal expression of any given protein. Grey box represents samples that were below the detection limit of the assay; B-D Representative cytokines and chemokines are reported (IL17A/F, MCP-1 and IL10 respectively). Each symbol is a distinct donor. Data are shown as mean±SD. Dots of the same shape represents samples polarised with the same cue; E-H Flow cytometry analysis of cell surface markers. Each symbol represents a donor. MFI ratio is calculated dividing the MFI of day5 or day7 polarised hMDMs by the MFI of day5 or day7 unpolarised hMDMs. The dotted line represents a ratio of 1: a ratio above 1 means upregulation in polarised vs unpolarised hMDMs. A ratio below 1 means downregulation.

[0265] FIG. 6: shows characterisation of various differentiation and polarisation protocols A Flow cytometry analysis of cell surface markers of hMDMs D5 and D7 polarised using a combination of rIL4 and rIL13 (20 ng/mL). Each symbol is an independent donor. Data are reported as RFI, calculated as MFI polarised/MFI unpolarised. The dotted line set at 1 on the graph represents therefore no modulation of the specific marker as compared to the unpolarised cells; B Flow cytometry analysis of CD206 in day5 hMDMs cultured in GMP cell culture bags at distinct densities. RFI is calculated dividing the MFI of the day5 hMDMs by the MFI of CD14+monocytes. Lines connect RFI values measured in hMDMs from a single donor; C Flow cytometry analysis of 25F9 in day5 hMDMs cultured in GMP cell culture bags at distinct densities. RFI is calculated dividing the MFI of the day5 hMDMs by the MFI of CD14+ monocytes. Lines connect RFI values measured in hMDMs from a single donor.

[0266] FIG. 7: shows characterisation of the day5 no feed protocol in GMP bags vs. standard plastic A Yield of the day5 no feed protocol in GMP bags using three distinct cell densities: 10.sup.6/cm.sup.2, 2×10.sup.6/cm.sup.2 and 3×10.sup.6/cm.sup.2. Each line connects data from a single donor, according to legend; B Viability of the hMDMs produced with the day5 no feed protocol in GMP bags using three distinct cell densities: 10.sup.6/cm.sup.2, 2×10.sup.6/cm.sup.2 and 3×10.sup.6/cm.sup.2. Each line connects data from a single donor, according to legend; C Yield of the day5 no feed protocol in GMP bags vs. standard plastic. The percentage (%) yield is calculated as: (Number of macrophages at harvest/number of macrophages plated)*100. D'Agostino and Pearson omnibus normality test was carried out, and then a non-parametric Mann Whitney test was applied; D Viability at the end of the day5 no feed protocol in GMP bags vs. standard plastic. The percentage (%) is calculated as: % of DRAQ-CD45+ macrophages in the single cell gate. D'Agostino and Pearson omnibus normality test was carried out, and then a non-parametric Mann Whitney test was applied; E Flow cytometry analysis of cell surface markers. Each symbol represents a donor. MFI ratio is calculated dividing the day5 MFI by the MFI of CD14+ monocytes at the start of the culture. The dotted line represents a ratio of 4, considered the minimal target ratio for CD206 and 25F9 to release the day7 macrophages for treatment. Data are analysed using t-test for unpaired data.

[0267] FIG. 8: shows injection of day5 hMDMs in mouse models of chronic and acute liver injury A Experimental design: NOD/SCID mice are injected twice a week for 12 weeks with CCl.sub.4to induce severe liver fibrosis. Day5 hMDMs are injected at the start of week 9, 10 and 11. Mice are culled one week later the last dose of macrophage therapy; B PSR staining is performed on the livers treated with day5 hMDMs or saline and stained areas are quantified. Each symbol represents a mouse. 6 to 10 10× fields/mouse are quantified. D'Agostino and Pearson omnibus normality test was carried out, and then a non-parametric Mann Whitney test was applied. p=0.12; C-D Dosage of circulating serum ALT (C) and bilirubin (D) at cull. Each dot represents a mouse. D'Agostino and Pearson omnibus normality test was carried out, followed by a one-tailed t-test for unpaired data with Welch's correction for samples with unequal variances. p non-significant for serum ALT, although a trend towards reduction in day5 hMDMs treated mice is reported (p=0.07); serum bilirubin was significantly reduced in day5 hMDMs treated mice at cull: *p<0.05 (p=0.02) for bilirubin; E Representative pictures of day5 hMDMs and saline treated livers. (10×, brightfield); F Experimental design: C57Bl/6 WT mice were starved for 14 h prior the injection of paracetamol (acetaminophen, APAP). Day5 hMDMs polarised to AAMs are injected 16 h post-APAP injection and mice culled 2 0h later (36 h post-AAP injection); G H&E staining is performed on the livers treated with day5 AAMs or saline and stained areas are quantified. Each dot represents a mouse. 6 to 10 10× fields/mouse are quantified. A Shapiro-Wilk normality test was applied, followed by a t-test for unpaired data was applied. p=0.13; H-I Dosage of circulating serum GLDH and AST. Each dot represents a mouse. A Shapiro-Wilk normality test was carried out, followed by a one-tailed t-test for unpaired data with Welch's correction for samples with unequal variances. For GLDH *p<0.05 (p=0.046), for AST p non-significant (p=0.263). J Representative haematoxylin and eosin stainings of livers treated with vehicles or day5 AAMs.

[0268] FIG. 9: shows the supporting data for the in vivo experiments carried out in FIG. 8. A Weight was monitored twice a week for 12 weeks during CCl.sub.4 treatment. Data mouse by mouse are plotted. We report in the vehicle treated mice and the hMDMs day5 treated mice. B Liver:weight ratio is not significantly different in vehicle vs. day5 hMDMs treated mice at the point of cull. C-E Serum levels of ALP (C), bilirubin (D) and albumin (E) in vehicle vs. day5 hMDMs treated mice at cull. F-H White Blood Cells (WBC, F), Red Blood Cells (RBC, G) and haematocrit (HCT, H) in vehicle vs. day5 hMDMs treated mice at cull. We reported a trend towards reduction in RBC and HCT in day5 hMDMs as compared to vehicle treated mice. I-J Serum IL6 (I) and IL10 (J) in vehicle vs. day5 hMDMs treated mice at cull. No difference are reported. A-J A Shapiro-Wilk normality test was carried out, followed by a one-tailed t-test for unpaired data with Welch's correction for samples with unequal variances. p<0.05 was considered statistically significant. K Liver:body weight ratio at cull 36 h after paracetamol (APAP) overdose in vehicle vs. day5 hMDMs treated mice at cull. L Weight drop at cull in vehicle vs. day5 hMDMs treated mice at cull. M-O Serum levels of ALP (M), bilirubin (N) and albumin (0) in vehicle vs. day5 hMDMs treated mice at cull. Bilirubin was significantly lower in day5 hMDMs treated as compared to vehicle treated mice. P-R White Blood Cells (WBC, P), Red Blood Cells (RBC, Q) and haematocrit (HCT, R) in vehicle vs. day5 hMDMs treated mice at cull. K-R A one-tailed t-test for unpaired data with Welch's correction for samples with unequal variances was carried out. p<0.05 was considered statistically significant. B-R Every dot represents a distinct mouse.

EXAMPLES

[0269] The present invention is further exemplified by the following examples. The examples are for illustrative purpose only and are not intended, nor should they be construed as limiting the invention in any manner.

[0270] Certain embodiments of the invention will now be demonstrated by way of the following non-limiting examples and with reference to the figures above.

Materials and Methods

GMP Human Monocyte-Derived Macrophages (hMDMs) Cell Culture

[0271] We isolated monocytes from a buffy coat product from a healthy volunteer sourced from the Scottish National Blood Transfusion Service (SNBTS) using a Ficoll gradient (GE Healthcare) followed by a magnetic column selection using CliniMACS CD14 Reagent (Miltenyi Biotec). We then matured monocytes for 1 to 7 days in culture in TexMACS without phenol red (Miltenyi Biotec) in the presence of 100 ng/mL GMP-graded recombinant human macrophage colony-stimulating factor (rhM-CSF) (R&D System, Biotechne). hMDMs day5 and day7 are cultured in 6 wells multi-well plate (Corning Costar) at a density of 2×10.sup.6/cm.sup.2. hMDMs d5 are also cultured in GMP cell culture bags (Miltenyi Biotec) using increasing concentrations: 1×10.sup.6/cm.sup.2, 2×10.sup.6/cm.sup.2 and 3×10.sup.6/cm.sup.2. hMDMs were fed at day 3 when matured for 7 days: briefly, half of the culture medium volume is added to each well/bag, supplemented with rhM-CSF at a final concentration of 100 ng/mL. Day5 and day7 hMDMs were counted using an automated counter (TC20, BioRad).

Flow Cytometry

[0272] hMDMs were harvested and spun at 300× g, 5 minutes, room temperature. hMDMs were re-suspended at a concentration of 10.sup.6/mL in PBS +2.5 mM EDTA+0.5% Albumin (PEA). Blocking was performed by incubating hMDMs in PEA with FcR blocking reagent (Miltenyi Biotec) 1:100 for 20 minutes at 4° C. Antibodies were added to the cell suspension at a dilution of 1:100 and incubated for 20 minutes at 4° C. (details of the antibodies used are reported in the table below).

TABLE-US-00001 TABLE 1 Antibodies ANTIGEN FLUOROPHORE CLONE SUPPLIER ORDERING CODE CD45 VB 5B1 Miltenyi Biotec 130-092-880 CD14 VB/PE TUK4 Miltenyi Biotec 130-091-242/130-113-152 CD206 FITC Miltenyi Biotec 130-095-131 25F9 APC 25F9 eBioscience 50-0115-42 CCR2 PE K036C2 BioLegend 357206 CD163 FITC Miltenyi Biotec BO-097-626 CD169 APC 7-239 BioLegend 346008 CD86 PE BU63 BioLegend 374206 MHC II FITC TU39 BioLegend 361705

[0273] Data were acquired using the Miltenyi Vyb flow cytometer and analysed with the MACS Quant software (Miltenyi Biotec).

V-Plex Cytokine Dosage

[0274] Cytokines in cell culture supernatants: cytokines were analysed using a V-PLEX Human Biomarker 54-Plex kit on a MESO Quickplex SQ 120 according to the manufacturers' instructions (Meso Scale Discovery). TGF-β1, TGF-β32 and TGF-β33 were analysed using the TGF-b V-plex kit on a MESO Quickplex SQ 120 according to the manufacturers' instructions (Meso Scale Discovery). Cytokines belonging to the IL17 family (IL17A/F, IL17B, IL17C, IL17F) were analysed using a V-plex kit on a MESO Quickplex SQ 120 according to the manufacturers' instructions (Meso Scale Discovery). 10 μL of supernatants were tested. Results are in pg/mL. Values are adjusted taking into consideration the distinct time in culture (hMDMs day7 secrete cytokines for 2 days more), higher dilution of cytokines in hMDMs day7 (they receive ⅓ more of medium as a result of the feed at day3) and average yield (yield of hMDMs d5 is higher than hMDMs d7).

Mouse Experiments

[0275] NOD CB17 Prkdc/.sup.SCID mice were supplied by Charles River and housed in individually ventilated cages in a sterile animal facility with a 10-14-hours dark/light cycle and free access to food and water. All procedures were performed in accordance with UK Home Office guidelines (Animals [Scientific Procedures] Act 1986). Chronic liver fibrosis was induced in adult male mice over a 12-week period by twice weekly intraperitoneal injections of carbon tetrachloride (CCl.sub.4) dissolved in sterile olive oil at a concentration of 0.2 mL/kg for the first week increasing to 0.4 mL/kg for the remaining 11 weeks. One day after the 18th CCl.sub.4 injection (9 weeks), mice were randomly allocated to receive either day5 hMDMs (n=10) or saline (vehicle, n=9) injections via tail vein. The intra-splenic route would have ensured maximal cell delivery, but it does not model the administration route used in the phase I MATCH trial (day7 hMDMs in patients with chronic liver fibrosis) (14). Day 5 hMDMs were suspended in sterile saline at a density of 5×10.sup.7 cells/mL and 0.1 mL was injected via a 30-gauge needle (Myjector 0.3 mL syringes, Terumo). Day5 hMDMs intravenous injection was repeated at week 10 and week 11. CCl.sub.4 administration continued for an additional week.

[0276] Wild-type C57BL/6J male mice (8-10 weeks old) were allowed to acclimatise for a minimum of one week in a clean animal facility. Prior to paracetamol (APAP) administration, mice were fasted at least 12 hours. Mice received a single injection (i.p.) of APAP (350 mg/kg) dissolved in warm saline between 20:00 and 22:00. Mice were left recovery until morning in a heated cabinet (27° C.); 16 h post-APAP overdose, they received hMDMs day5 polarised towards alternatively activated phenotype (AAMs) using rhIL4+rhIL13 (20 ng/mL) and rhM-CSF (100 ng/mL) for 24 h.

[0277] All mice were culled at the indicated time points using anaesthesia overdose followed by cervical dislocation as confirmatory method. Organs and blood were retrieved, processed and stored for further analysis: liver left lobe was snap frozen and stored at −80° C.; the other liver lobes were fixed in formalin 10% for 8 h and then included in paraffin blocks; kidneys, spleen, heart and lungs were fixed in formalin 10% for 8 h and then included in paraffin blocks; blood was collected in Eppendorf, left to sediment for 8 h and then spun at 10000× g for 10 minutes at room temperature to obtain serum, to be stored at −80° C.; blood collected in EDTA-coated tubes (Microvette CB300, Sarstedt) were used to collect 30 μL of full blood to use for the analysis of the haematological parameters using the CellTac machine (Nihon Kohden).

Liver Function Tests on Sera

[0278] Serum chemistry was performed by measurement of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin, and serum albumin. ALT was measured using a commercial kit (Alpha Laboratories Ltd). AST and ALP were determined by a commercial kit (Randox Laboratories). Total bilirubin was determined by the acid diazo method described by Pearlman and Lee (20) using a commercial kit (Alpha Laboratories Ltd). Mouse serum albumin measurements were determined using a commercial serum albumin kit (Alpha Laboratories Ltd). All kits were adapted for use on a Cobas Fara centrifugal analyzer (Roche Diagnostics Ltd). For all assays, intra-run precision was CV<4%. In some experiments, assays were run on plasma samples with the exception of ALP activity.

[0279] Here below it is reported a table to highlight which parameters are more helpful to evaluate liver damage in acute and chronic models (+++=extremely helpful; ++=very helpful; +=helpful; ±=moderately helpful; −=not helpful).

TABLE-US-00002 TABLE 2 Liver Damage Parameters ALT AST GLDH ALP BILIRUBIN Chronic CCl.sub.4 + + − ± +++ Acute APAP O.D. ++ ++ +++ ± ++

Histological Analysis

[0280] Haematoxylin and eosin (H&E) and picrosirius red (PSR) staining were performed according to standard protocols. Morphometric pixel analysis to quantify histological staining was performed. For necrosis and fibrosis quantification respectively, H&E and PSR stained section were scanned to create a single image with Polaris slide scanner (Perkin Elmer) A second scan on the same machine was performed to obtain multi-spectral image acquisition on 10 to 15 fields/slide at 10× magnification. Multi-spectral images were analysed using the Trainable WEKA Segmentation mode using the InForm software (Perkin Elmer).

Statistical Analysis

[0281] All data are expressed as mean±standard deviation (SD). The number of replicates is indicated in each figure and each replicate represent a biological rather than an experimental replicate. Data are analysed and graphs are generated with GraphPad Prism version 8 (GraphPad Software, Inc, USA). Statistic test has been chosen depending on the biological question behind the experiment. Briefly we used Student's t-test, one- or two-way ANOVA followed by an appropriate post-hoc test. The test used is stated in each figure legend. P<0.05 is considered statistically significant.

[0282] For all in vitro experiments a two-sided test is considered. All data were tested for normal distribution and equal variance before performing any statistical analysis using Prism v8.

[0283] We have performed a power calculation for the number of mice to use in the studies on the chronic CCl.sub.4 model based on the data available from previous studies on the level of ALT (indicating liver damage) at 12 weeks of CCl.sub.4 treatment. We have assumed a mu1 of 100 for 12 week-CCl.sub.4 mice treated with hMDMs and a mu2 of 200 for 12 week-CCl.sub.4 mice treated with vehicle (saline), with a sigma of 50. We have set the power desired at 0.80 assuming a statistical significance at the threshold of 0.05. The power calculation returned an n=6. This is the minimal number of mice used in each experiment. We had no available data at the time of the experiment for the APAP overdose mice treated with hMDMs day5 polarised to AAMs for 24 h. We treated the present experiment as a pilot and we plan to expand the treated cohort in the future.

[0284] For all in vivo experiments a one-sided test is considered, as we are testing the hypothesis that human macrophages reduce necrosis and fibrosis in APAP overdose and CCl.sub.4 models, respectively. All data were tested for normal distribution and equal variance before performing any statistical analysis using Prism v8. Specific tests used are indicated in each legend to figure. Power calculation has been performed using the free online tool available at http://www.stat.ubc.ca.

Results

[0285] The inventive differentiation protocol delivers an increase in yield and viability of macrophages as compared to the current gold standard day7 protocol

[0286] Monocytes cultured for five days (herein referred to as day5) in TexMacs medium with 100 ng/mL show similar characteristics to those cultured for seven days with a re-feed at day three (current gold standard protocol, herein referred to as day7) as observed using brightfield microscopy (FIG. 1A). The day5 protocol shows a strong trend towards a better yield (FIG. 1B, p=0.06), similar viability and metabolism (FIG. 1C-E) as compared to the day7 protocol. One of the main concerns in delivering cell therapy by i.v. infusion is the potential for cells to clump once in the bloodstream. We tested whether the day5 protocol raised the ability of the hMDMs to adhere to a plastic surface. We observed that the day5 hMDMs show a trend towards less adhesion (FIG. 1F, p=0.1875) as compared to the day7 hMDMs.

[0287] The inventive differentiation protocol leads to the production of mature, fully functional GMP-compliant macrophages

[0288] A major concern in shortening the protocol and eliminating the feed is the generation of partially mature, non-fully functional macrophages. We compared the secretion profile of the day5 vs. day7 hMDMs using a highly sensitive 54-plex platform to test the cell culture supernatants at the end of the differentiation protocol. We report herein the results of the dosage of some cytokines, chemokines and growth factors involved in acute and chronic liver disease (FIG. 2A) (a correction factor that takes into account differences in time of culture and yield has been applied). Day5 hMDMs show reduced levels of IL17F and TGFβ1 (Transforming Growth Factor β1), both previously associated with a detrimental effect during liver cirrhosis (21-24). Day5 hMDMs do not upregulate cytokines such as IL6, IL8 and TNFα (Tumour Necrosis Factor α), thereby confirming that these cells are safe to be used as cell therapy as they are unlikely to cause a cytokine storm upon injection (see also Table 5).

[0289] A major issue with using a shorter protocol without re-feeding could be the generation of hMDMs lacking some of the cell surface receptors typical of mature macrophages, such as CD206 (Mannose Receptor), CD163 (Hemoglobin-Aptoglobin Scavenger Receptor) and CD169 (sialoadhesin). Macrophages generated with a shorter protocol may also have aberrant expression of antigen presenting molecule (e.g. CD86 and MHC Class II) and may fail to downregulate CCR2 (C-C Chemokine Receptor type 2, or CD192). We show herein that day5 hMDMs have a cell surface marker expression similar to day7 hMDMs (FIG. 2B and Table 1). Currently, the minimal standard to define a ‘mature’ macrophage generated for clinical use (as per MHRA-compliant clinical protocol) is a 5-fold increase in the expression of CD206 and 25F9 (a marker associated with macrophage maturation). Both our protocols meet this condition (dotted line in FIG. 2B). 25F9 is not strongly upregulated in our preparations as compared to the currently used clinical product because our starting material consists of monocytes from peripheral blood mononuclear cell preparations taken from healthy donor blood donations, which have a higher 25F9 expression than monocytes from leukapheresis (i.e. those used to generate the day7 product for clinical use).

[0290] One of the desired function of macrophages once transferred in a patient is phagocytosis: Phagocytosis contributes to dead or dying cell clearance, restoration of the liver's barrier function against bacteria of gut origin, and conversion of macrophage phenotype from pro-inflammatory to pro-restorative. In fact, one of the main concerns in reducing the cell culture time and eliminating the feeding is that the hMDMs obtained could be less efficient at performing phagocytosis. Using a live imaging approach to measure phagocytosis of zymosan A-coated beads by day5 vs day7 hMDMs we demonstrated that the hMDMs generated with the two protocols are comparable in terms of phagocytic capacity (FIG. 2C and Table 4).

TABLE-US-00003 TABLE 3 RFI of cell surface markers in hMDMs day5 no-feed and day7 standard protocol marker DAY5 av DAY5 SD n DAY7 av DAY7 SD n CD14 3.526405 2.236156 18 2.044955 1.316308 14 CD206 54.95115 28.16548 18 55.68434 31.11817 14 25F9 4.20196 2.513642 18 2.697895 1.591471 14 CD163 23.63787 11.53435 13 20.3617 9.779279 13 CD169 11.61222 9.765346 13 16.2687 14.03805 13 CCR2 0.232945 0.2091234 13 0.2619833 0.1339491 13 CD86 6.808384 4.273312 10 5.580 6.354599 7 MHC Class II 5.21494 3.227986 10 10.47571 16.34271 7

TABLE-US-00004 TABLE 4 Alex488 cytoplasmic MFI as a measure of phagocytosis in day5 no-feed and day7 standard protocol hMDMs DAY5 DAY7 TIME MEAN SD n MEAN SD n 0 0.000 0.000 7 0.000 0.000 6 10 −1.441384 2.895393 7 −1.2243 4.132898 6 20 0.3100007 4.042008 7 0.03003117 6.089865 6 30 3.872093 6.089653 7 3.304473 7.824904 6 40 8.268787 8.024744 7 7.496515 9.180511 6 50 13.12365 10.32989 7 11.64244 11.01407 6 60 17.68846 12.61675 7 16.20512 12.25722 6 70 22.09463 15.05047 7 20.88522 13.55408 6 80 25.95883 17.14326 7 25.60332 14.70255 6 90 29.56796 19.36675 7 30.29026 15.81196 6 100 33.09268 21.40685 7 34.29896 16.95813 6 110 36.85434 23.43145 7 38.40984 18.2587 6 120 40.00526 25.15578 7 42.76797 19.05064 6 130 43.19535 26.48693 7 46.53022 20.54206 6 140 46.29604 28.4563 7 50.02634 21.74133 6

TABLE-US-00005 TABLE 5 Secretion profile of day5 (D5) vs. day7 (D7) standard protocol hMDMs: t-test or non- D5 D7 parametric test Average SD Average SD D5 vs D7 IL17 family IL17A/F 26.88 25.14 37.09 28.00 0.0427 IL17B 19.32 16.02 21.12 16.27 0.4478 IL17C 76.88 65.18 98.03 63.89 0.1052 IL17F 146.76 110.29 210.91 128.66 0.0182 TGFb family TGFb1 43871.69 2290.83 44755.05 3038.96 0.0444 TGFb2 6066.80 322.57 6108.69 368.11 0.5375 TGFb3 13.06 1.84 14.64 0.76 0.0563 Vacsular SAA 137.30 196.15 109.55 160.79 0.6398 injury CRP 712.06 499.76 629.62 204.40 0.6499 sVCAM1 6412.58 1050.65 6887.30 1843.64 0.2543 sICAM1 30.58 18.44 17.13 11.79 0.0651 Angiogenesis VEGF-A 183.49 126.81 135.16 131.12 0.3665 family VEGF-D 0.00 0.00 3.39 5.21 0.1360 PIGF 0.28 0.40 0.30 0.25 0.9102 bFGF 0.53 0.34 0.61 0.41 0.6795 VEGFR1 124.45 75.09 171.11 102.55 0.0109 cytokines IL3 85471.30 13505.88 86694.60 14221.14 0.0596 IL6 0.57 0.40 0.59 0.42 0.9215 IL1RA 10710.74 9054.36 12113.01 10312.49 0.2939 IL9 103.70 55.62 128.67 51.26 0.0106 TNFa 2.54 1.73 1.87 1.06 0.0708 chemokines IL8 2.27 3.10 5.50 9.73 0.4878 IP10 81.41 41.38 105.20 61.46 0.2369 MCP1 3610.78 3491.17 7055.61 4318.41 0.0631 MCP4 5.18 9.06 24.16 16.85 0.0549 TARC 9.54 9.66 10.29 6.34 0.8298

[0291] We tested whether the presence of a small percentage of human serum type AB (0.5%) and/or the usage of another cell culture medium (AimV) could further improve yield and viability of day5 hMDMs. As shown in FIG. 3, we failed to identify any differences in yield and viability (FIGS. 3A-B and E). We also observed that AimV-cultured cells were in general more prone to form clumps in cell culture, therefore raising a major safety concern around its use. We concluded that the usage of human serum does not improve yield, viability or metabolism (FIG. 3C-D) of day5 hMDMs. We also decided against the use of AimV in further experiment because of the cell clumping concern.

[0292] Characterisation of the day-by-day differentiation of human monocytes into macrophages using the inventive protocol

[0293] Despite the most common finding in literature is that macrophages need to be cultured for around a week, we have shown that day5 hMDMs are fully mature and functional. We reasoned that perhaps some characteristic of mature macrophages could be present even before the fifth day of culture. We therefore performed a day-by-day flow cytometry of cell surface markers on monocytes from two distinct donors (FIG. 4). We cultured the monocytes for either five or seven days, with or without feeding. We stopped the analysis of the unfed cells at day5. We were able to show that CCR2 is downregulated as early as day one after the start of the culture, and it is at levels similar to those of mature macrophages from day 2 onwards (FIG. 4F). Markers such as CD206, 25F9 and CD169 increase over time, and peak at day 6, to then decline by day 7 (FIG. 4B, C, E). An upregulation of these markers are seen as early as 3 days in culture (FIG. 4B, C, E).This reinforces our hypothesis that day7 hMDMs maybe be too “spent” to perform optimal therapeutic functions once transferred in vivo. Our analysis also unveiled that hMDMs cultured in TexMacs supplemented with 100 ng/mL MCSF have a cell surface marker expression compatible with mature cells from day 4, regardless of the presence or absence of feeding. However, the expression of some of the cell surface marker at day 4 is not stabilised yet as shown by the variable levels of CD163 (FIG. 4D) and by fluctuating levels of CD86 (FIG. 4G). The present inventors have identified that cells at day 4 are juvenile cells and therefore more amenable to processing such as cryopreservation and transfection. The analysis of the viability during the cell culture reveal a drop at day2 and day6 (FIG. 4E). The drop at day2 explains the lower-than-100% yield that we routinely observe in our experiments. The drop at day6 could justify the trend to a much lower yield in the day7 vs. day5 protocol, as reported in FIG. 1B.

The Macrophages of the Invention are Mature and Able to Respond to Inflammatory Cues

[0294] One of the key features of macrophages is their ability to respond to inflammatory cues. It has long been described that in vivo macrophages are a highly heterogeneous population, which acquire distinct polarisation depending on the microenvironment. Hyper-responsiveness to inflammatory cues is one of the concerns when using a day5 vs a day 7 hMDMs differentiation protocol. We therefore went on to demonstrate that our day5 hMDMs is safe and does not generate cells that could potentially cause a cytokine storm once injected in a patient with an ongoing acute or chronic liver inflammation.

[0295] To this end, we collected supernatants from day5 hMDMs and day7 hMDMs after 24 h stimulation with IFNγ, IFNγ plus LPS, IL4 plus IL13 and IL10. We performed the same 54-plex analysis reported in FIG. 2 followed by a clustering analysis using the online tool Morpheus. Data suggest that IFNγ and IFNγ plus LPS-stimulated macrophages clustered together, regardless of whether the unpolarised macrophages were differentiated using a day5 or day7 protocol. Interestingly, IL4 plus IL13 and IL10 macrophages, which should share more anti-inflammatory, pro-restorative properties, clustered together based on the donor they came from, rather than the cell culture protocol to obtain the unpolarised macrophages (FIG. 5A). A closer look at some important cytokines and chemokines in acute and chronic liver disease (IL17A/F, MCP1 and IL10) further confirms that no significant differences are detectable in the way day5 unpolarised macrophages react to inflammatory stimuli (FIG. 5B-D). These results suggest that the day5 hMDMs differentiation protocol leads to the generation of cells that do not over-react to inflammatory cues and are therefore unlikely to cause a cytokine storm once injected in a patient with acute or chronic liver disease.

[0296] Polarised macrophages can be beneficial in a number of pathological setting, and to favour the success of other cell therapies. Therefore, there could be an interest in producing polarised macrophages starting from the day5 or day7 unpolarised hMDMs. We verified by flow cytometry whether day5 and day7 hMDMs have a similar cell surface marker expression following polarisation. We calculated the RFI by dividing the MFI of the polarised for the MFI of the unpolarised hMDMs: a RFI>1 means upregulation; a RFI<1 means downregulation (FIG. 5E-H). The main feature of LPS-stimulated macrophages is the upregulation of antigen presenting molecule such the MHC II molecules (Major Histocompatibility Complex Class II). Both day7 and day5-polarised hMDMs showed an increase of their MHC II molecules (FIG. 5F). Following stimulation with IFNγ, day7-polarised hMDMs showed an unusual upregulation of CD206, a scavenger receptor that should be induced by the IL4/1L13 stimuli. Conversely, IFNγ day5-polarised hMDMs failed to show this upregulation (FIG. 5E). IL10 stimulation led to the upregulation of CD163 and the downregulation of CD86 and MHCII in both day5 and day7 hMDMs, as expected (FIG. 5G). However, when we used the IL4/1L13 combination we fail to see a significant upregulation of CD206 in day5-polarised hMDMs (FIG. 7A). We reasoned that adding fresh MCSF could have better supported polarisation. We therefore run a comparison between day5 and day7-polarised hMDMs, in the presence or in the absence of MCSF. The addition of MCSF resulted in CD206 upregulation in IL4/1L13 day5 polarised hMDMs (FIG. 5H). These data reinforce the concept that day5 hMDMs are mature macrophages, able to normally respond to inflammatory stimuli, and that can be used as starting material for the production of polarised human macrophages to use as cell therapy.

[0297] The Inventive differentiation protocol leads to the production of mature, fully functional macrophages using GMP-graded cell culture bags

[0298] hMDMs for clinical use cannot be grown using cell culture-treated plastic. Normally, hMDMs for clinical use are cultured using GMP-compliant cell culture bag. Therefore, we sought to validate our day5 no feed differentiation protocol using the above-mentioned cell culture bag as a support.

[0299] We reasoned that perhaps a critical factor for the growth in GMP-graded cell culture bag is cell density: We tested our day5 hMDMs differentiation protocol using the density used in plastic (2×10.sup.6/cm.sup.2), a lower (1×10.sup.6/cm.sup.2) and a higher (3×10.sup.6/cm.sup.2) density. We also pondered that macrophages may benefit from a more crowded environment at the start of the differentiation process, to then benefit from less dense culture conditions. Therefore, we went on to culture monocyte/macrophages at 2×10.sup.6/cm.sup.2 till day3, to then dilute them at 1×10.sup.6/cm.sup.2 for the last two days of culture. Best results in terms of yield were obtained using 3×10.sup.6/cm.sup.2 (FIG. 7A). Viability was very variable at 1×10.sup.6/cm.sup.2. 2×10.sup.6/cm.sup.2 and 3×10.sup.6/cm.sup.2 cell density showed a consistent viability of 90% or more in all donors analysed (FIG. 7B). We then compared the cell surface expression of mature macrophage markers in day5 hMDMs cultured in cell culture bags at distinct densities. Day5 hMDMs from all donor showed high expression of CD206; only one donor (donor SP105) showed levels of 25F9 non compatible with the current release criteria (RFI≥4). Variation between protocols were minimal (FIG. 6B, C). Diluting the macrophages during the last two days of culture did not offer a significant advantage as compared to the 2×10.sup.6/cm.sup.2 cell density (not shown). The slightly worst results on viability obtained with the 1×10.sup.6/cm.sup.2 suggest that macrophages need paracrine signals for their differentiation and growth. We concluded that a density of 2×10.sup.6/cm.sup.2 or 3×10.sup.6/cm.sup.2 can be used in the future for the production of day5 hMDMs for clinical use.

[0300] We then ought to confirm that the functional results obtained using hMDMs cultured on plastics were translatable to hMDMs cultured in cell culture bags. To this end we carried on a comparison of the yield, viability and cell surface markers of the day5 hMDMs culture in plastic or GMP-graded cell culture bags at a density of 2×10.sup.6/cm.sup.2. Cells cultured in bags show similar yield and viability to those cultured using tissue culture-treated plastic plates (FIG. 7C, D). The expression of cell surface markers is similar between the d5 hMDMs cultured in bags and in plastic (FIG. 7E and Table 6).

TABLE-US-00006 TABLE 6 RFI of cell surface markers in hMDMs day5 no feed protocol in GMP vs. standard plastic DAY5 DAY5 plastic DAY5 GMP DAY5 GMP marker plastic av SD n bag av bag SD n CD14 3.526405 2.236156 18 4.84125 3.85 8 CD206 54.95115 28.16548 18 71.50875 98.17 8 25F9 4.20196 2.513642 18 6.3325 5.73 8 CD163 23.63787 11.53435 13 27.24 7.420505 3 CD169 11.61222 9.765346 13 5.753 3.05896 3 CCR2 0.232945 0.2091234 13 0.1467 0.040414 3 CD86 6.808384 4.273312 10 10.11 6.819318 3 MHC Class II 5.21494 3.227986 10 12.77 6.472328 3

Macrophages of the Invention are Safe to Inject in Mouse Models of Acute and Chronic Liver Injury

[0301] To definitely prove that our day5 hMDMs are a suitable product for cell therapy of acute and chronic liver injury we needed to test their safety in mouse models. We also aimed to show signs of efficacy of the therapy. To this end we induced liver cirrhosis by injecting carbon tetrachloride (CCl.sub.4) into immunodeficient mice twice a week for 12 weeks. We injected 10.sup.6 day5 hMDMs or vehicle (saline) at the beginning of week 9,10 and 11. We culled the mice one week later (FIG. 8A). The mice did not show any adverse event at the time of injection of the cell therapy, nor during the week prior to culling. Mice in the treated group did not lose weight and their weight trends over time and liver:body weight ratio at cull were comparable to controls (FIG. 9A, B). Haematological parameters such as white blood cells (WBC), red blood cells (RBC) and haematocrit (HCT) were comparable in the two groups of mice, too (FIG. 9F, G, H), thereby excluding severe adverse effects on the bone marrow of the treated mice. There is a significant trend to have a reduce HCT in the treated mice, a trend potentially relevant as the therapy restores normal HCT levels for adult male NOD/SCID mice (pink dashed line in FIG. 9H). Cytokines such as IL6 and IL10 were at comparable levels in the plasma of the two groups of mice (FIG. 9I, J), confirming that the day5 hMDMs are safe to inject and do not trigger a cytokine storm when injected in a mouse experiencing chronic inflammation.

[0302] Further, we assessed whether we could detect signs of efficacy. To this end we performed liver function tests (LFTs) on the sera of treated and control mice. No difference in the levels of ALT was detected, although we reported a trend do decreased AST (FIG. 8C, FIG. 9D). No differences in the levels of ALP and albumin were detected either (FIG. 9C, E). Remarkably, bilirubin was significantly reduced in the sera of mice treated with hMDMs day5 (FIG. 8D). ALT and AST circulating levels can be influenced by various factors, including macrophage phagocytosis: in fact, they can be internalised by macrophages via this process. Therefore, an analysis of fibrosis in the liver tissue is needed to assess efficacy. We performed picrosirius red (PSR) staining on the liver sections from the treated and the control group to quantify fibrosis: treated mice showed a strong trend towards reduced fibrosis (FIG. 8B, E). Immunodeficient mice lack the adaptive arm of immunity, a key player in liver regeneration. They are therefore partially impaired in taking advantage of the paracrine effect of macrophage cell therapy. Furthermore, the immune response to damage is not the same magnitude of wild type mice, thereby partially impairing the microenvironment-macrophage cross-talk, a process crucial to mediate the therapeutic effect of macrophages. Therefore, a trend towards reduction both in circulating AST and liver fibrosis, and a significant reduction in circulating bilirubin is a remarkable result. In conclusion, data suggest that injecting day5 hMDMs in models of chronic liver disease is safe and partially efficacious in reducing liver fibrosis.

[0303] Previous work in the lab suggested that alternatively activated macrophages (IL4/IL13 polarised, herein referred to as AAMs) are useful to contain acute liver injury by paracetamol (acetaminophen, APAP) overdose and to promote liver regeneration in mice (Starkey Lewis P J et al., J Hep, 2020). We sought to repeat the same data using our day5 hMDMs polarised using IL4/IL13 plus MCSF, as described in FIG. 7 as product for cell therapy of APAP overdose. APAP overdose was induced in C57Bl/6 immunocompetent mice (FIG. 8F). We previously demonstrated that healthy immunocompetent mice receiving day7 hMDMs do not show signs of rejection or toxicity up to one week after the injection of the cells (not shown). This prompted us to use an immunocompetent mouse for our safety and efficacy test as this overcomes the limitations of immunocompromised mice listed above. The treated mice showed a trend towards an increased liver:body weight ratio and increased weight loss (FIG. 9K, L), which could potentially raise safety concerns. However, haematological parameters such as WBC, RBC and HCT were comparable in the two groups of mice (AAMs day5 treated vs. vehicle treated) (FIG. 9P, Q, R), thereby excluding an ongoing toxicity at bone marrow level. Further, ALP and Albumin were comparable in the two groups of mice (FIG. 9M, O), excluding an exacerbation of the liver damage by the AAMs day5 therapy. Conversely, mice showed significantly reduced levels of circulating GLDH (FIG. 8H), a sensitive marker for necrotic tissue damage. Mice also showed smaller necrotic area when treated with AAMs day5 as compared to livers of untreated mice, as assessed by image quantification of H&E sections (FIG. 8G, J). A further indication of efficacy is given by the reduction in circulating bilirubin observed in the treated mice (FIG. 9N).

[0304] In conclusion, our data shows that we have set up a protocol for generating GMP-graded hMDMs in a cheaper and faster way as compared to current standard. This result has been achieved by combining two approaches: reducing the cell culture time and eliminating the feeding step. The use of the optimal cell density, a serum-free T-cell medium, with the support of rMCSF, guarantees a product that has superior qualities to the day7 hMDMs; the current gold standard in the field. In particular, day5 hMDMs shows a less pro-inflammatory and less pro-fibrogenic secretion profile (lower levels of IL17F and TGFβ in the cell culture supernatants respectively). Despite literature in the field suggesting that day7 is the optimal length of culture to produce mature hMDMs, we showed that our day5 hMDMs product is comparable in terms of cell surface marker expression and phagocytosis to the day7 hMDMs product. Furthermore, the day5 hMDMs differentiation protocol shows a better yield as compared to the day7 hMDMs protocol. Day5 hMDMs furthermore proved safe and efficacious in mouse models of APAP overdose (acute liver injury); they also proved safe and partially efficacious in mouse models of liver cirrhosis (chronic liver injury).

[0305] In view of the above, it will be appreciated that the present disclosure also relates to the following clauses:

[0306] Clause 1: An in vitro method of producing macrophages comprising: [0307] (a) Culturing monocytes in medium for 4-5 days to produce macrophages, wherein the medium comprises one or more growth factors to stimulate macrophage production;

[0308] And wherein step (a) takes place entirely in the same medium.

[0309] Clause 2: The method according to clause 1, wherein the monocytes are cultured for 5 days.

[0310] Clause 3: The method according to any one of clauses 1 to 2, wherein the method does not comprise re-feeding or replacing medium.

[0311] Clause 4: The method according to any one of clauses 1 to 3, wherein the monocytes are seeded at a density of between 1×10.sup.6 cells/cm.sup.2 up to 1×10.sup.7 cells/cm.sup.2.

[0312] Clause 5: The method according to any one of clauses 1 to 4, wherein the medium is selected from X-Vivo 10, TexMACS, AIMv, RPMI, DMEM, and DMEM/F12.

[0313] Clause 6: The method according to clause 5 wherein the medium is a T-cell medium, preferably TexMACS.

[0314] Clause 7: The method according to any one of clauses 1 to 6, wherein the medium comprises one or more factors selected from the CSF family, preferably M-CSF.

[0315] Clause 8: The method according to any one of clauses 1 to 7, wherein the medium contains M-CSF at a concentration of between 25-150 ng/mL.

[0316] Clause 9: The method according to any one of clauses 1 to 8, wherein the monocytes are human, and the macrophages are human monocyte derived macrophages (hMDMs).

[0317] Clause 10: The method according to any one of clauses 1 to 9, wherein the monocytes are derived from human blood, preferably the buffy coat of human blood, preferably from the PBMC fraction of human blood.

[0318] Clause 11: The method according to any one of clauses 1 to 10, wherein the method further comprises a step of polarisation of the macrophages produced in step (a), preferably into M1-like or M2-like macrophages.

[0319] Clause 12: The method according to clause 11, wherein polarising factors are added to the medium, preferably M1 or M2 polarising factors.

[0320] Clause 13: The method according to clause 12, wherein the M1 polarizing factors are selected from: GM-CSF, IFNγ, and TLR agonists such as LPS; and the M2 polarizing factors are selected from: IL10, IL4, IL13, and poly(I:C).

[0321] Clause 14: The method according to any one of clauses 1 to 13, wherein the method is GMP-compliant.

[0322] Clause 15: The method according to one of clauses 1 to 14, wherein the yield of mature macrophages is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%.

[0323] Clause 16: A macrophage produced by the method according to any of one of clauses 1 to 15.

[0324] Clause 17: An ex vivo generated macrophage having a pro-regenerative phenotype.

[0325] Clause 18: The macrophage according to any one of clauses 16 or 17, wherein the macrophage has increased expression of one or more pro-regenerative cytokines.

[0326] Clause 19: The macrophage according to any of one clauses 16 to 18, wherein the macrophage has increased expression of one or more anti-inflammatory cytokines and/or reduced expression of one or more inflammatory cytokines, and/or increased expression of one or more anti-fibrogenic cytokines, and/or reduced expression of one or more fibrogenic cytokines.

[0327] Clause 20: The macrophage according to any of one clauses 16 to 19 wherein the macrophage has reduced expression of one or more of the following cytokines: IL1, IL12, IL17 (A, B, C, F), IL18, TNFα, IFNγ, preferably IL17F.

[0328] Clause 21: The macrophage according to any of one clauses 16 to 20, wherein the macrophage has reduced expression of one or more of the following cytokines: IL4, IL13, PDGF, TGFβ (1, 2, 3), preferably TGFβ1.

[0329] Clause 22: The macrophage according to any of one clauses 16 to 21, wherein the macrophage expresses mature cell surface markers, preferably CCR2−, CD14+, CD206+, CD163+, CD169+, 25F9+, and CD86+.

[0330] Clause 23: A population of macrophages according to any of one clauses 16 to 22.

[0331] Clause 24: A composition comprising a population of macrophages according to clause 23.

[0332] Clause 25: A macrophage, population or composition according to any of one clauses 16 to 24 for use as a medicament.

[0333] Clause 26: A macrophage, population, or composition according to any of one clauses 16 to 24 for use in the treatment of liver disease or injury.

[0334] Clause 27: A macrophage, population, or composition for use according to clause 26, wherein the liver disease is acute or chronic.

[0335] Clause 28: A macrophage, population or composition for use according to clause 27, wherein the acute liver disease is a drug overdose preferably APAP overdose, and wherein the chronic liver disease is liver cirrhosis.

[0336] Clause 29: A population or composition according to clause 23 or 24, wherein the macrophages express an average of less than 200 pg/mL of IL17F, preferably less than 190 pg/mL of IL17F, preferably less than 180 pg/mL of IL17F, preferably less than 170 pg/mL of IL17F, preferably less than 160 pg/mL of IL17F, preferably 150 pg/mL or less of IL17F.

[0337] Clause 30: A population or composition according to clauses 23, 24 or 29, wherein the macrophages express an average of less than 45000 pg/mL of TGFβ, preferably less than 44000 pg/mL, preferably less than 43000 pg/mL, preferably less than 42000 pg/mL, preferably less than 41000 pg/mL, preferably 40000 pg/mL or less of TGFβ.

[0338] Clause 31: A population or composition according to clauses 23, 24, 29 or 30, wherein the macrophages express on average more than 50 pg/mL VEGFR1, preferably more than 100 pg/mL of VEGFR1, preferably more than 120 pg/mL of VEGFR1, preferably more than 140 pg/mL VEGFR1, preferably more than 160 pg/mL VEGFR1, preferably more than 170 pg/mL of VEGFR1.

[0339] Clause 32: A population or composition according to any of clauses 23, 24 and 29-31, wherein the macrophages express on average less than 500 pg/mL of IL9, preferably less than 300 pg/mL of IL9, preferably less than 200 pg/mL IL9, preferably less than 180 pg/mL IL9, preferably less than 160 pg/mL IL9, preferably less than 140 pg/mL IL9, preferably less than 130 pg/mL of IL9.

[0340] Clause 33: A population or composition according to any of clauses 23, 24 and 29-32, wherein the macrophages possess an anti-inflammatory and anti-fibrogenic phenotype, wherein the macrophages express on average less than 200 pg/mL of IL17F and less than 45000 pg/mL of TGFβ.

[0341] Clause 34: A population or composition according to any one of clauses 23, 24 and 29-33 in which the macrophages have an anti-inflammatory and anti-fibrogenic phenotype, wherein the macrophages express on average around 150 pg/mL of IL17F and around 40000 pg/mL of TGFβ.

[0342] Clause 35: A macrophage, population or composition according to any of clauses 16-24 which respond to inflammatory stimuli such as one or more of: IFNγ, IL10, IL4, IL13, and LPS.

[0343] Clause 36: A cell culture bag comprising a macrophage of any of clauses 1-22, a population according to clause 23, or a composition according to clause 24.

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EQUIVALENTS

[0368] Those skilled in the art will recognise, or be able to ascertain using no more than routine experimentation, equivalents of the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims. Any combination of the embodiments disclosed in the any plurality of the dependent claims or Examples is contemplated to be within the scope of the disclosure.

INCORPORATION BY REFERENCE

[0369] The disclosure of each and every patent, patent application publication, and scientific publication referred to herein is specifically incorporated herein by reference in its entirety, as are the contents of its Figures.