CRYOPRESERVING MACROPHAGES

Abstract

The present invention relates to a method of improving the viability of macrophages subjected to cryopreservation, particularly for macrophages which are to be used in therapy, wherein the method comprises a step of maintaining the macrophages at a temperature of 2-12° C. for at least 30 minutes during either freezing or thawing procedures. Following the holding step during cooling, a cooling rate of 1 to 5° C. is used until the macrophages in a medium are frozen. For thawing the macrophages, a warming rate of 1 to 5° C. per minute is used until a temperature of 35-37° C. is reached. The present invention further relates to the cryopreserved macrophages, and the thawed macrophages produced by such methods. The technique may provide macrophages that are GMP-compliant and have a viability of at least 60%.

Claims

1. A method of improving the viability of macrophages subjected to cryopreservation, wherein said method comprises the following steps during freezing and/or thawing of the macrophages: (a) bringing the macrophages in a medium to a temperature of 2-12° C., and maintaining this temperature for at least 30 minutes; followed by either: (b) cooling of the macrophages in a medium at a rate of 1 to 5° C. until the macrophages in the medium are frozen; or (c) warming of the macrophages in the medium at a rate of 1 to 5° C. per minute until a temperature of 35-37° C. is reached.

2. A method according to claim 1 wherein the method is for freezing macrophages, the method comprising: (a) placing macrophages in medium; (b) cooling the medium containing macrophages of step (a) to a temperature of between about 2-12° C. and maintaining the cooled medium at a temperature of between about 2-12° C. for a period of at least 30 minutes; and (c) freezing the medium containing macrophages of step (b) at a cooling rate of 1-5° C. per minute.

3. A method according to claim 1 wherein the method is for thawing cryopreserved macrophages, wherein the cryopreserved macrophages are in a medium, the method comprising: (a) warming cryopreserved macrophages to a temperature of between about 2-12° C. and maintaining the medium at a temperature of between about 2-12° C. for a period of at least 30 minutes; and (b) warming the cryopreserved macrophages of step (a) at a warming rate of 1-5° C. per minute until a temperature of 35-37° C. is reached.

4. The method of claim 3 further comprising one or more additional steps: (i) diluting the cryopreserved macrophages of step (a) in medium; and/or (ii) maintaining the macrophages of step (b) at a temperature of about 37° C. for at least 30 minutes.

5. A method for improving the viability of macrophages subjected to cryopreservation, wherein said method comprises the following steps: (a) placing macrophages in medium; (b) cooling the medium containing macrophages of step (a) to a temperature of between about 2-12° C. and maintaining the cooled medium at a temperature of between about 2-12° C. for a period of at least 30 minutes; (c) freezing the medium containing macrophages of step (b) at a cooling rate of 1-5° C. per minute; (d) warming cryopreserved macrophages to a temperature of between about 2-12° C. and maintaining the medium at a temperature of between about 2-12° C. for a period of at least 30 minutes; and (e) warming the cryopreserved macrophages of step (a) at a warming rate of 1-5° C. per minute until a temperature of 35-37° C. is reached.

6. The method of claim 5 further comprising one or more additional steps: (i) diluting the cryopreserved macrophages of step (d) in medium; and/or maintaining the macrophages of step (e) at a temperature of about 37° C. for at least 30 minutes.

7. The method of any preceding claim wherein the macrophages are isolated macrophages or macrophages produced in vitro.

8. The method of any preceding claim wherein the macrophages are present in the medium at a concentration of at least 5×10.sup.6 cells/mL, optionally at least 1×10.sup.7 cells/mL.

9. The method of any preceding claim wherein the macrophages are present in a medium containing a cryoprotectant.

10. The method of claim 9 wherein said medium is selected from CS2, CS5, CS10, albumin and DMSO, PRIME-XV FreezIS, FREEZEstem, or STEM-CELLBANKER, either alone or in combination with PlasmaLtye, TexMACS, human albumin.

11. The method of claim 4 or 6, wherein the medium for diluting contains rhMCSF (recombinant human macrophage colony stimulating factor), optionally at a concentration of between 50-150 ng/mL.

12. The method of any preceding claim wherein the method is GMP compliant.

13. Cryopreserved macrophages produced according to the method of claim 1 or 2.

14. Thawed macrophages produced according to the method of claims 1 or 3 to 12.

15. A cryopreserved therapeutic composition comprising a population of cryopreserved macrophages according to claim 13, wherein upon thawing, said macrophages have a viability of at least 60%.

16. The composition of claim 15, said viability being at least 60% after at least 3 months of cryopreservation, preferably at least 6 months of cryopreservation, preferably at least 1 year of cryopreservation.

17. The composition of claim 15 or 16 wherein the cryopreserved macrophages are thawed according to the method of any one of claims 1 or 3 to 12.

18. Cryopreserved macrophages according to claim 13 or thawed macrophages according to claim 14, for use as a medicament.

19. Cryopreserved macrophages according to claim 13 or thawed macrophages according to claim 14, for use in the treatment of a liver disease.

20. Cryopreserved or thawed macrophages for use according to claim 19, wherein the liver disease is liver cirrhosis.

Description

EXAMPLES

[0347] 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.

1. Materials and Methods

[0348] GMP Human Monocyte-Derived Macrophages (hMDMs) Cell Culture

[0349] 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 were fed at day 3 when matured for 7 days: briefly, half of the culture medium volume is added to each well, supplemented with rhM-CSF at a final concentration of 100 ng/mL. Day5 and day7 hMDMs were counted using an automated counter (TC20, BioRad).

Freezing of Human Monocyte-Derived Macrophages (hMDMs)

[0350] hMDMs day5 and hMDMs day7 were re-suspended in the following medium: Albumin+10% DMSO, Albumin+20% DMSO, CryoStore 10 (CS10), and Prime XV. hMDMs day5 and day7 were re-suspended in the various freezing medium at the following concentrations: 1×10.sup.6/mL, 5×10.sup.6/mL, 10×10.sup.6/mL, 20×10.sup.6/mL, 50×10.sup.6/m L. hMDMs day5 and day7 were cooled down prior to freezing for either 30 mins or 60′mins by placing each vial at 4° C. in a Mr Frosty container, which progressively lower the temperature at a rate of about 1° C./min. hMDMs day5 and day7 were frozen at −80° C. in Mr Frosty for a period of time between 2 weeks and 6 months.

Thawing of Human Monocyte-Derived Macrophages (hMDMs)

[0351] In the first experiment, hMDMs day5 and day7 were thawed, diluted 1:10 in GMP-graded TexMACS (Miltenyi Biotec) and spun at 300×g, 5 mins at 4° C. They were then re-suspended in 10 mL of GMP-graded TexMACS and cultured for 2 h in 6-multi well plates (Cornig-Costar) at 37° C., 5% CO.sub.2 prior to viability analysis as described below.

[0352] In all the other experiments, hMDMs day5 and day7 were diluted 1:10 in cold (4 C. to 15 C.) GMP-graded TexMACS (Miltenyi Biotec). The cell suspension was directly cultured for 2 h in 6-multi well plates (Cornig-Costar) at , 5% CO.sub.2 prior to viability analysis. In selected experiments, the cell suspension was progressively warmed up by placing the diluted cell suspension at 4° C. for 1 h prior to culture at 37° C., 5% CO.sub.2, 5% CO.sub.2. In selected experiments, rhMCSF at a concentration of 100 ng/mL (R&D System, Bio-techne) was added to the GMP-graded TexMACS. After the culture at 37° C., 5% CO.sub.2, the cell suspension was subjected to viability analysis as described below.

Viability Analysis

[0353] After 2 h to 4 h of cell culture at 37° C., 5% CO.sub.2, hMDMs day5 and day7 were harvested and counted using a TC20 cell counter (BioRad). 10.sup.5 to 10.sup.6 cells were placed in a flow cytometry-compatible 5 mL tube (Falcon). The cell suspension was then analysed using a Milteny Vibe flow cytometer to set the baseline fluorescence. DRAQ7 (Abcam) was then added to the cell suspension at 1:500 dilution. The cell suspension was immediately analysed using a Miltenyi Vibe flow cytometer. Data were analysed and results obtained with the MACS Quant software (Miltenyi Biotec).

Phagocytosis Assay

[0354] 10.sup.5 hMDMs day5 and day7 post-thaw were placed in a 5 mL flow cytometry-compatible tube (Falcon) in a volume of 100 uL of PBS+2.5 mM EDTA. 100 μL of green zymosan-A coated pHrodo beads (Invitrogen, Life Technologies) were added to the cell suspension. pHrodo beads were prepared following the manufacturer's instructions. After 1 h of beads-cell co-culture, 1 mL of PBS+2.5 mM EDTA was added to each tube. Tubes were then spun at 200×g, 5 mins at 4° C. The supernatant was then eliminated and cells were re-suspended 1004 of PBS+2.5 mM EDTA+1% human albumin (PEA). We prepared two tubes per sample, in order to have a single staining for the pHrodo beads, and a double staining with a macrophage-specific marker. An anti-mouse CD14 antibody conjugated with VioBlue (VB) was added to the cell suspension at a concentration of 1:100 to identify macrophages. After 20 mins of incubation, the excess of antibody was washed away by adding 1 mL of PEA and by spinning the cell suspension as indicated above. The cells were re-suspended in 500 μL of PEA, and DRAQ7 (Abcam) was added at a 1:500 dilution immediately prior to analysis. The cell suspension was analysed using a Miltenyi Vibe flow cytometer. Results were analysed with the MACS Quant software (Miltenyi Biotec).

Mouse Experiments

[0355] 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 further 10 weeks. One day after the 18th CCl.sub.4 injection (9 weeks), mice were randomly allocated to receive either day 5 cryopreserved (CP) 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) (19). 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 CP hMDMs intravenous injection was repeated at week 10 and week 11. 0.2 mL/kg CCl.sub.4 administration continued for an additional week.

[0356] 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 304 of full blood to use for the analysis of the haematological parameters using the CelITac machine (Nihon Kohden).

Liver Function Tests on Sera

[0357] 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.

V-Plex Cytokine Dosage

[0358] Cytokines in the sera of treated mice were analysed using a V-PLEX Mouse Biomarker 10-Plex kit on a MESO Quickplex SQ 120 according to the manufacturers' instructions (Meso Scale Discovery). 10 uL of serum pre-diluted 1:5 were tested. Results are in pg/mL. The results presented herein take into consideration the above mentioned dilution factor.

Histological Analysis

[0359] Picrosirius red (PSR) staining was performed according to standard protocols. Morphometric pixel analysis to quantify histological staining was performed. For fibrosis quantification 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

[0360] 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.

[0361] 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.

[0362] We performed 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 mug 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.

[0363] For all in vivo experiments a one-sided test is considered, as we are testing the hypothesis that human macrophages reduce fibrosis in CCI.sub.4 models. 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.0 bc.ca

2. Results

[0364] In order to optimise a GMP-graded protocol to freeze and thaw mature human monocyte-derived macrophages (hMDMs) we first tested the procedure reported in FIG. 1A. Cooling down time before freezing and choice of freezing medium are two key steps to optimise when setting up a cryopreservation protocol: the freezing process alters the cytoplasmic composition, particularly with respect to pH and salt concentrations (21, 22); moreover, cryopreservation agents such as DMSO may both protect and cause damage to the cells (23-26). In the first set of experiments, we tested two cool down times (30 minutes and 60 minutes) at 4° C. prior to transferring the hMDMs at −80° C. in Mr Frosty to guarantee a 1° C./min cooling rate. hMDMs were frozen in a standard medium containing human albumin (Alb)+20% DMSO. hMDMs matured for either five (D5) or seven (D7) days using GMP-graded recombinant hMCSF were used in this experiment. hMDMs were frozen at distinct densities, namely 1×10.sup.6, 5×10.sup.6 and 10×10.sup.6/mL. hMDMs were thawed, spun, counted and analysed by flow cytometry to establish viability. We set 60% viability as the minimal threshold to deem the protocol viable for translation into a therapeutic setting. hMDMs cooled down both for 30 min or 60 min prior to freezing showed insufficient viability at thaw, with the 10×10.sup.6/mL concentration performing slightly better than lower concentrations (FIGS. 1B-C). The calculated yield was good both for the 30 min and the 60 min cool down time. hMDMs showed a preference for a higher concentration at the point of freezing in this case too (FIGS. 1D-E).

[0365] We then reasoned that spinning hMDMs just after thaw could have been the cause of the observed drop in viability: The cell's plasma membrane is weakened by the cryopreservation process, and spinning could cause further damage, and possibly leading the cell to cell death. We therefore repeated the same freezing procedure, followed by a thawing phase in which hMDMs were left for 2 h in the incubator in complete medium (TexMACS+rhMCSF) prior to analysis of viability by flow cytometry. In this set of experiments, we also tried a higher concentration of hMDMs at point of freezing (20×10.sup.6/mL). We tested both D5 and D7 hMDMs from the same donor (FIG. 1F). We observed an improved viability for all the conditions tested. Particularly, we reached the desired 60% minimum viability with the D5 hMDMs cooled down for 60 mins and frozen at a concentration of 20×10.sup.6/mL (FIG. 1G). The D5 hMDMs offered also a better yield as compared to their D7 counterparts (FIG. 1H).

[0366] To further improve the protocol, we decided to test the two highest concentrations (10×10.sup.6/mL and 20×10.sup.6/mL) with various freezing medium: Alb+20% DMSO and CS10, a GMP-graded, 10% DMSO-containing medium. CS10 is normally recommended for other cryopreservation-sensitive cell types (induced pluripotent stem cells and mesenchymal stem cells) https://www.stemcell.com/cryostor-cs10.htmL). We therefore reasoned that it could be apt to efficiently cryopreserve hMDMs. We kept the 60 mins cooling down time as it gave the best results in the previous experiments (FIG. 2A). When we analysed the viability at the point of thaw, CS10 was the best performing medium, with viability up to 80% for D5 hMDMs and up to 70% for D7 hMDMs (FIGS. 2B-C). Yield was better with CS10 for D5 hMDMs frozen at 20×10.sup.6/mL (FIG. 2D). D7 hMDMs gave mixed results in terms of yield (FIG. 2E), highlighting that D5 hMDMs are the best product to bring forward to be successfully cryopreserved.

[0367] DMSO is one of the most used cryopreservation agents, however a GM P-graded DMSO-free medium is available (Prime-XV). Therefore, we decided to compare viability obtained with CS10 freezing medium and with Prime-XV. Further, in the previous experiment CS10 was compared to our home-made Alb+20% DMSO. To exclude that the key to increased viability was the 10% DMSO we compared viability obtained with CS10 and with Alb+10% DMSO. Finally, we wanted to explore the possibility that a gradient of temperature at thawing could benefit viability: every preparation was split at the time of thawing and placed either at 4° C. for 1 h, before being transferred into the incubator at 37° C., or directly into the incubator (FIG. 2F). CS10 delivered the best viability, both when hMDMs were thawed at 4° C. and at 37° C. Prime XV was the next best performing medium when hMDMs were thawed at 4° C. (FIG. 2G). We therefore concluded that (i) 10% DMSO is not sufficient to guarantee optimal D5 hMDMs viability, (ii) CS10 is the best performing medium and (iii) Prime XV is the next best alternative, but hMDMs have to be thawed at 4° C. if this medium is used.

[0368] As discussed previously, the cryopreservation property can alter the intracellular concentration of salts and pH (21, 22): therefore, when evaluating a novel cryopreservation protocol, it is important to functionally test the cryopreserved cells at the point of thaw. One of the most important functions of macrophages is phagocytosis, a process that involves progressive acidification of intracellular compartments to digest the phagocytic cargo and that in turn shapes the phenotype and function of macrophages during inflammation (27-30). We therefore evaluated the ability of the thawed hMDMs of phagocytosing zymosan-A coated beads. hMDMs cryopreserved with any protocols showed a good phagocytic capacity, with hMDMs frozen in CS10 and Prime-XV performing better than hMDMs frozen in Alb+10% DMSO (FIGS. 2H-F). Finally, we evaluated hMDMs viability post-phagocytosis. hMDMs frozen in CS10 showed the best post-phagocytosis viability, especially when thawed at 4° C. (FIG. 2J). We therefore concluded that thawing at 4° C. makes hMDMs more resilient to demanding tasks. The drop in viability compared to the pre-phagocytosis condition is normally observed in this assay, during which hMDMs are read at room temperature using a flow cytometer over the span of few hours.

[0369] We further validated our choice to use a progressive temperature increase at thawing by comparing viability and phagocytosis of a sample of hMDMs cryopreserved for three months (FIG. 3A). The analysis of viability at the point of thaw confirmed that thawing with a progressive increase of temperature is the most efficient method (40% viability when thawing at 37° C. vs 80% viability when thawing at 4° C., then at 37° C.) (FIG. 3B). Phagocytosis was similar for hMDMs thawed with or without progressive increase in temperature (FIG. 3C). hMDMs are cultured in TexMACS medium supplemented with 100 ng/mL of GMP-graded recombinant human MSCF (rhMCSF, also known as CSF1). The use of rhMCSF in the medium at thawing could or not be a key aspect of the protocol. Therefore, in order to define the best minimal conditions to cryopreserve hMDMs, we compared preparations from three distinct donors: half of each preparation was thawed with the usual protocol in the presence or in the absence of rhMCSF in the thawing medium (FIG. 3D). No difference was observed when viability post-thaw was analysed (FIG. 3E). It is possible that the presence of rhMCSF helps hMDMs during thawing when they have been frozen for longer periods of time. We tested the effects of rhMCSF in the thawing medium in hMDMs cryopreserved for 3 months. We are also aware that other cell types are frozen in 66% DMSO in some cell therapy protocols currently tested in clinical trials. Therefore, we compared the effect of 100% CS10 vs. 66% CS10 on yield and viability at the point of thawing (FIG. 3F). Yield was similar for hMDMs cryopreserved in 100% and 66% CS10 (FIG. 3G). Viability was slightly better for hMDMs cryopreserved in 100% CS10 vs. hMDMs cryopreserved in 66%. The effect of rhMCSF was unclear, for some donors improving, for some donors worsening viability (FIG. 3H).

[0370] We have therefore established that the ideal protocol to cryopreserve hMDMs entails cooling down hMDMs prior to freezing for 60 minutes, using CS10 or Prime-XV as freezing medium, and a concentration of 20×10.sup.6 or higher. The ideal thawing process entails a progressive increase in temperature, and a rest time for hMDMs of at least 2 h in incubator at 37° C. prior to use. The use of rhMCSF in the thawing medium (TexMACS) is dispensable but may be used in some cases (e.g. thawing hMDMs after three or more months of cryopreservation).

[0371] The next step has been to validate the protocol for long time freezing (five to six months) (FIG. 4A). We analysed yield and viability of a prep of D5 and D7 hMDMs from a single donor cryopreserved for 5 months. Yield and viability were excellent both for D5 and D7 hMDMs (above 60% viability for both, FIGS. 4B-C). We also tested D5 and D7 hMDMs from a single donor which have been cryopreserved for 6 months. Yield was good both for D5 and D7 hMDMs. However, a drop below the 60% mark was noticed for the D5 hMDMs (FIGS. 4D-E). We also measure the phagocytic capacity of hMDMs D5 and D7 after 6 months of cryopreservation. Both D5 and D7 hMDMs were able to perform phagocytosis, with D5 hMDMs performing better than D7 hMDMs (21% vs. 14%) (FIG. 4F).

[0372] Macrophages polarise to distinct phenotypes depending on the cytokines cues they receive (31-35). Murine polarised macrophages have been used in several models of disease (2, 6, 36-39). Here we sought to prove that our cryopreservation protocol works for human GMP-graded polarised hMDMs. To this end, we produced alternatively activated macrophages (AAMs) by stimulating D5 hMDMs with a combination of IL4 and IL13 for 24 h. We then froze them using CS10 at three increasing concentrations (10×10.sup.6/mL, 20×10.sup.6/mL, 50×10.sup.6/mL) (FIG. 4G). Yield at 10×106/mL is poor (around 20%), although viability reaches the 60% threshold. Much better yield and viability are achieved using a freezing concentration of 20×10.sup.6/mL and 50×10.sup.6/mL (FIG. 4H-I). We analysed the phagocytic capacity of AAMs from three distinct donors frozen at a concentration of 20×10.sup.6/mL; AAMs with all three donors were able to phagocytose, albeit at different levels depending on the donor (FIG. 4J). We therefore concluded that our protocol can be utilised to cryopreserve polarised hMDMs, and that polarised macrophages have a preference for higher concentrations at the point of freezing.

[0373] We reasoned that the present cryopreservation protocol will be first used to cryopreserve unpolarised macrophages from cirrhotic patients to perform autologous cell therapy in the future stages of our clinical trial ((19) and clinical trial number ISRCTN 10368050). Hence, we proceeded to validate our cryopreservation protocol on D7 hMDMs from cirrhotic patients that exit the production line for quality control. We took D7 hMDMs from two patients (M2 and M3) and we froze them in 100% CS10 at increasing concentration (10×10.sup.6/mL, 20×10.sup.6/mL, 50×10.sup.6/mL) and we tested yield, viability and phagocytosis at the point of thaw after two weeks of cryopreservation. We also froze D7 hMDMs from patients M3 in 66% CS10 and we compared yield, viability and phagocytosis at the point of thaw with D7 hMDMs from the same patient frozen in 100% CS10 (FIG. 5A). Patient M2 showed a drop of yield and viability at a freezing concentration of 20×10.sup.6/m L. The best yield and viability were achieved when a freezing concentration of 50×10.sup.6/mL was used (FIGS. 5B-C). Phagocytosis was progressively more efficient moving from a freezing concentration of 10×10.sup.6/mL to 50×10.sup.6/mL (FIG. 5D). Conversely, D7 hMDMs from patients M3 showed a peak yield of almost 80% when a freezing concentration of 20×10.sup.6/mL was used (FIG. 5E). Viability at the point of thawing progressively increased with the increase of the freezing concentration and peaked at 50×106/mL (FIG. 5F). Phagocytosis at thawing was similar for 20×10.sup.6/mL and 50×10.sup.6/mL concentrations, and slightly higher for 10×10.sup.6/mL (FIG. 5G). We concluded that hMDMs from cirrhotic patients need higher concentration at the point of freezing to preserve high viability. Yield and phagocytic ability vary, and are possibly more patient-dependent than method-dependent. Using 66% CS10 at the point of freezing increases the yield for D7 hMDMs frozen at a concentration of 50×10.sup.6/mL, while decreases the yield for the same cells frozen at a concentration of 20×10.sup.6/mL (FIG. 5H). Viability at the point of thaw was similar for D7 hMDMs frozen in 100% CS10 and 66% CS10 at both 20×10.sup.6/mL and 50×10.sup.6/mL (FIG. 5L). Phagocytosis was higher for D7 hMDMs frozen at a concentration of 20×10.sup.6/mL in 66% CS10, and similar for all the other conditions tested (FIG. 5J). We concluded that our protocol effectively cryopreserves hMDMs from cirrhotic patients, both when using 100% and 66% CS10, and a high freezing concentration.

[0374] We further the validation of our protocol by measuring yield, viability and phagocytosis of hMDMs from cirrhotic patients cryopreserved for long period of time (4 months). hMDMs were frozen at 10×10.sup.6/mL, 20×10.sup.6/mL and 50×10.sup.6/mL, and thawed in the presence or in the absence of rhMCSF in the thawing medium (TexMACS). We also compared yield and viability for hMDMs cryopreserved in 100% or 66% CS10 (FIG. 6A). We analysed hMDMs from patient M2 after four months of cryopreservation, and we recorded an excellent yield (nearing 100%) for all the freezing concentrations (FIG. 6B). Satisfactory viability (60%) was reached only for hMDMs frozen at a concentration of 50×10.sup.6/mL. The addition of rhMCSF in the thawing medium slightly improved viability for the 10×10.sup.6/mL freezing concentration (FIG. 6C). Phagocytosis was similar for all freezing concentrations tested and in line with previously obtained results (FIG. 6D). hMDMs from patient M3 were frozen at a concentration of 20×10.sup.6/mL in either 100% or 66% CS10. When thawed after four months the yield was around 60% both when hMDMs were frozen in 100% and 66% CS10 (FIG. 6E). Viability was slightly higher when 66% CS10 was used as freezing medium. The addition of rhMCSF to the thawing medium improved viability, especially when 66% CS10 was used as freezing medium, pushing viability above the 60% target (FIG. 6F). Phagocytosis was similar for hMDMs frozen in 100% and 66% CS10 (FIG. 6F), and addition of rhMCSF to the thawing medium failed to change the hMDMs phagocytic ability either (FIG. 6G).

[0375] We therefore concluded that long term cryopreservation of cirrhotic hMDMs is feasible with our protocol. Ideally, a freezing concentration between 10×10.sup.6/mL and 50×10.sup.6/mL should be used, with a preference for higher concentrations. Freezing can be performed with 100% or 66% CS10, and the addition of rhMCSF in the thawing medium (TexMACS) could be beneficial to increase viability.

[0376] Mouse models of liver cirrhosis triggered by reiterative CCl.sub.4-induced hepatocyte injury are a useful tool to test the safety and efficacy of cell therapy product. The induction phase of liver cirrhosis commonly last 4 to 12 weeks, depending on the extent of fibrosis desired (40-42). We envisage our cell therapy being used in cases of advanced fibrosis therefore we chose to treat our mice for 12 weeks with CCl.sub.4. Testing a cryopreserved macrophage-based cell therapy product requires a xenotransplant of human cell into mice. To avoid rejection, we opted to use immunodeficient mice. However, because these mice lack an appropriate immune response to liver fibrosis, they are unlikely to benefit from the paracrine effect of macrophage cell therapy on the mouse own immune response. In the present experiment, we inject 1×10.sup.6 cryopreserved hMDMs at week 9, 10 and 11 of CCl.sub.4 treatment. Control mice are injected with an equivalent volume of saline only (vehicle). Mice are culled at week 12 and blood and organs collected for further analysis (FIG. 7A). Histological analysis of the quantity of fibrosis in the liver by PSR staining and quantification revealed an average decrease in fibrosis of 13% in cryopreserved hMDMs treated vs. saline treated mice (FIG. 7B). Sera analysis confirmed a positive effect of the cryopreserved hMDMs cell therapy: Results show a trend towards a decrease in liver enzymes ALT and AST (FIGS. 7C-D) and, more importantly, a significant reduction in bilirubin circulating levels (FIG. 7E), one of the main factors in monitoring the results of macrophage cell therapy in the clinic (19). No change in circulating GLDH and Albumin was noted (FIGS. 7G-H). Despite the cell therapy being carried out on an immunodeficient background, we were able to detect some signal of an anti-inflammatory activity following the injection of our cryopreserved hMDMs cell therapy: we show herein a decrease in circulating IL6 and an increase in circulating IL10 (FIGS. 7I-J).

[0377] In conclusion, we have shown that we have establish a novel protocol to cryopreserve functional hMDMs for cell therapy, we have validated the protocol in cirrhotic patients, and we have shown that the cryopreserved cell therapy product is effective at reducing fibrosis in a mouse model of liver cirrhosis. The protocol entails a cool down time in freezing medium prior to transfer at −80° C. of at least 30 minutes, preferentially up to 60 minutes. The freezing medium of choice should be CS10, either at 100% or lower concentration down to 66%. Alternatively, Prime XV should be considered as DMSO-free freezing medium option. hMDMs prefer to be cryopreserved at high concentrations (from 10×10.sup.6 to over 50×10.sup.6/mL). Using rhMCSF at the point of thaw delivers mixed result and may be consider when hMDMs are cryopreserved for long period of times (≥3 months). hMDMs have a preference for being thawed with a progressive increase in temperature: hMDMs should be left at 4° C. for at least 1 h prior to be diluted in cell culture medium (TexMACS) and left to recover in incubator (37° C., 5% CO.sub.2) for at least another hour. hMDMs are then ready for functional assays (e.g. phagocytosis) and/or for transplant as cell therapy.

Clauses of the Invention:

[0378] A. A method of cryopreserving macrophages, the method comprising: [0379] (a) Placing macrophages in medium; [0380] (b) Cooling the medium of step (a) to a temperature of between about 2-22° C. at a cooling rate of 1-5° C. per minute, and maintaining the cooled medium at a temperature of between about 2-22° C. for a period of at least 30 minutes; and [0381] (c) Freezing the medium of step (b). [0382] B. The method according to clause A, wherein the cooled medium is maintained at a temperature of between about 2-22° C. for a period of between 30 to 120 minutes, preferably for a period of between 30 to 60 minutes. [0383] C. The method of clauses A or B, wherein the medium is cooled to a temperature of between about 2-20° C., preferably between about 2-18° C., preferably between about 2-16° C., preferably between about 2-14° C., preferably between about 2-12° C., preferably between about 2-10° C., preferably between about 2-8° C., and maintained at the same temperature, preferably the medium is cooled to a temperature of between about 4-6° C. and maintained at the same temperature. [0384] D. The method of any preceding clause, wherein the cooling rate during cooling of the medium is selected from: 1° C., 1.1° C., 1.2° C., 1.3° C., 1.4° C., 1.5° C., 1.6° C., 1.7° C., 1.8° C., 1.9° C., 2° C., 2.1° C., 2.2° C., 2.3° C., 2.4° C., 2.5° C., 2.6° C., 2.7° C., 2.8° C., 2.9° C., 3° C., 3.1° C., 3.2° C., 3.3° C., 3.4° C., 3.5° C., 3.6° C., 3.7° C., 3.8° C., 3.9° C., 4° C., 4.1° C., 4.2° C., 4.3° C., 4.4° C., 4.5° C., 4.6° C., 4.7° C., 4.8° C., 4.9° C., and 5° C. per minute. [0385] E. The method of clause D, wherein the cooling rate during cooling of the medium is about 1° C. per minute. [0386] F. The method according to any preceding clause, wherein the medium is selected from one or more of: human albumin, PlasmaLyte, TexMACS, DMSO CS10, and PrimeXV. [0387] G. The method according to clause F, wherein the medium is a mixture of CS10 and TexMACS. [0388] H. The method according to clause G, wherein the medium comprises about 66% CS10 and 33% TexMACS. [0389] I. The method according to any preceding clause, wherein the macrophages are present in the medium at a cell concentration of between 1×10.sup.6-1×10.sup.8/mL. [0390] J. The method according to clause I, wherein the macrophages are present in the medium at a cell concentration of between 2×10.sup.7-5×10.sup.7/mL. [0391] K. The method according to any preceding clause wherein the freezing of the medium takes place at a temperature selected from −70° C., −71° C., −72° C., −73° C., −74° C., −75° C., −76° C., −77° C., −78° C., −79° C., −80° C., −81° C., −82° C., −83° C., −84° C., −85° C., −86° C., −87° C., −88° C., −89° C., −90° C., −91° C., −92° C., −93° C., −94° C., −95° C., −96° C., −97° C., −98° C., and −100° C. [0392] L. A method of thawing cryopreserved macrophages, the method comprising: [0393] (a) Warming cryopreserved macrophages to a temperature of between about 4-10° C., and maintaining the macrophages at a temperature of between about 4-10° C. for a first period, wherein the first period is at least 30 minutes; [0394] (b) Diluting the cryopreserved macrophages of step (a) in medium; and [0395] (c) Warming the cryopreserved macrophages of step (b) to a temperature of about 37° C., and maintaining the macrophages at a temperature of about 37° C. for a second period, wherein the second period is at least 30 minutes. [0396] M. The method of clause L, wherein the first and second periods are between 1 to 5 hours. [0397] N. The method of clause M, wherein the first period is about 1 hour. [0398] O. The method of clause M or N, wherein the second period is about 2 hours. [0399] P. The method of any of clauses L-O wherein the warming rate during warming of the medium in step (a) or step (c) is selected from: 1° C., 1.1° C., 1.2° C., 1.3° C., 1.4° C., 1.5° C., 1.6° C., 1.7° C., 1.8° C., 1.9° C., 2° C., 2.1° C., 2.2° C., 2.3° C., 2.4° C., 2.5° C., 2.6° C., 2.7° C., 2.8° C., 2.9° C., 3° C., 3.1° C., 3.2° C., 3.3° C., 3.4° C., 3.5° C., 3.6° C., 3.7° C., 3.8° C., 3.9° C., 4° C., 4.1° C., 4.2° C., 4.3° C., 4.4° C., 4.5° C., 4.6° C., 4.7° C., 4.8° C., 4.9° C., and 5° C. per minute, preferably the warming rate during warming of the medium in step (a) or step (c) is about 1° C. per minute. [0400] Q. The method of clause P, wherein step (a) comprises warming the cryopreserved macrophages to a temperature of between about 4-9° C., preferably between 4-8° C., preferably between 4-7° C., preferably between 4-6° C., and maintaining the macrophages at this temperature. [0401] R. The method according to any of clauses L-Q, wherein the medium for diluting contains MCSF (macrophage colony stimulating factor). [0402] S. The method according to clause R, wherein the MCSF is at a concentration of between 50-150 ng/mL in the medium. [0403] T. The method according to any of clauses L-S, wherein the macrophages are diluted in the medium by a factor of between 2 to 20. [0404] U. The method according to any preceding clause wherein the method is GMP compliant. [0405] V. A method according to any preceding clause, wherein the macrophages are human monocyte derived macrophages (hMDMs). [0406] W. A method comprising the steps of the method of cryopreserving macrophages according to any of clauses A-K, and the steps of the method of thawing macrophages according to any of clauses L-T. [0407] X. Cryopreserved macrophages produced by the method of any one of clauses A-K. [0408] Y. Thawed macrophages produced by the method of any one of clauses L-W. [0409] Z. A cryopreserved therapeutic composition comprising a population of cryopreserved macrophages according to clause X , wherein upon thawing, said macrophages have a viability of at least 60%. [0410] AA. The composition of clause Z, wherein the macrophages are at a concentration of 10×10.sup.6 -50×10.sup.6/mL, [0411] BB. The composition of clause Z or AA, said viability being at least 60% after at least 3 months of cryopreservation, preferably at least 6 months of cryopreservation, preferably at least 1 year of cryopreservation. [0412] CC. The composition of clause Z wherein the cryopreserved macrophages are thawed according to the method of clause L. [0413] DD. Cryopreserved macrophages according to clause X or thawed macrophages according to clause Y, for use as a medicament. [0414] EE. Cryopreserved macrophages according to clause X or thawed macrophages according to clause Y, for use in the treatment of a liver disease. [0415] FF. Cryopreserved or thawed macrophages for use according to clause EE, wherein the liver disease is liver cirrhosis.

REFERENCES

[0416] 1. T. Suzuki et al., Pulmonary macrophage transplantation therapy. Nature 514, 450-454 (2014). [0417] 2. M. Bacci et al., Macrophages are alternatively activated in patients with endometriosis and required for growth and vascularization of lesions in a mouse model of disease. Am J Pathol 175, 547-556 (2009). [0418] 3. A. Capobianco et al., Proangiogenic Tie2(+) macrophages infiltrate human and murine endometriotic lesions and dictate their growth in a mouse model of the disease. Am J Pathol 179, 2651-2659 (2011). [0419] 4. D. Danon, M. A. Kowatch, G. S. Roth, Promotion of wound repair in old mice by local injection of macrophages. Proc Natl Acad Sci USA 86, 2018-2020 (1989). [0420] 5. D. Danon et al., Treatment of human ulcers by application of macrophages prepared from a blood unit. Exp Gerontol 32, 633-641 (1997). [0421] 6. M. Z. Zhang et al., IL-4/IL-13-mediated polarization of renal macrophages/dendritic cells to an M2a phenotype is essential for recovery from acute kidney injury. Kidney Int 91, 375-386 (2017). [0422] 7. J. K. Moore et al., Phenotypic and functional characterization of macrophages with therapeutic potential generated from human cirrhotic monocytes in a cohort study. Cytotherapy 17, 1604-1616 (2015). [0423] 8. J. K. Moore et al., Patients with the worst outcomes after paracetamol (acetaminophen)-induced liver failure have an early monocytopenia. Aliment Pharmacol Ther, (2016). [0424] 9. J. A. Thomas et al., Macrophage therapy for murine liver fibrosis recruits host effector cells improving fibrosis, regeneration, and function. Hepatology 53, 2003-2015 (2011). [0425] 10. M. S. Hu et al., Delivery of monocyte lineage cells in a biomimetic scaffold enhances tissue repair. JCI Insight 2, (2017). [0426] 11. L. Arnold et al., Inflammatory monocytes recruited after skeletal muscle injury switch into antiinflammatory macrophages to support myogenesis. J Exp Med 204, 1057-1069 (2007). [0427] 12. F. W. van der Meulen, M. Reiss, E. A. Stricker, E. van Elven, A. E. von dem Borne, Cryopreservation of human monocytes. Cryobiology 18, 337-343 (1981). [0428] 13. M. J. ASHWOOD-SMITH, Preservation of mouse bone marrow at −79 degrees C. with dimethyl sulphoxide. Nature 190, 1204-1205 (1961). [0429] 14. H. E. Broxmeyer, S. Cooper, High-efficiency recovery of immature haematopoietic progenitor cells with extensive proliferative capacity from human cord blood cryopreserved for 10 years. Clin Exp Immunol 107 Suppl 1, 45-53 (1997). [0430] 15. H. E. Broxmeyer et al., High-efficiency recovery of functional hematopoietic progenitor and stem cells from human cord blood cryopreserved for 15 years. Proc Natl Acad Sci USA 100, 645-650 (2003). [0431] 16. M. Mandl, S. Schmitz, C. Weber, M. Hristov, Characterization of the CD14++CD16+ monocyte population in human bone marrow. PLoS One 9, e112140 (2014). [0432] 17. A. Delforge, E. Ronge-Collard, P. Stryckmans, T. Spiro, M. A. Malarme, Granulocyte-macrophage progenitor cell preservation at 4 degrees C. Br J Haematol 53, 49-54 (1983). [0433] 18. P. Walbrun et al., Characterization of rat and human Kupffer cells after cryopreservation. Cryobiology 54, 164-172 (2007). [0434] 19. F. Moroni et al., Safety profile of autologous macrophage therapy for liver cirrhosis. Nat Med, (2019). [0435] 20. F. C. Pearlman, R. T. Lee, Detection and measurement of total bilirubin in serum, with use of surfactants as solubilizing agents. Clin Chem 20, 447-453 (1974). [0436] 21. P. Mazur, Freezing of living cells: mechanisms and implications. Am J Physiol 247, C125-142 (1984). [0437] 22. P. Mazur, K. W. Cole, Influence of cell concentration on the contribution of unfrozen fraction and salt concentration to the survival of slowly frozen human erythrocytes. Cryobiology 22, 509-536 (1985). [0438] 23. J. K. SHERMAN, DIMETHYL SULFOXIDE AS A PROTECTIVE AGENT DURING FREEZING AND THAWING OF HUMAN SPERMATOZOA. Proc Soc Exp Biol Med 117, 261-264 (1964). [0439] 24. J. K. Sherman, Low temperature research on spermatozoa and eggs. Cryobiology 1, 103-129 (1964). [0440] 25. J. K. Sherman, Pretreatment with protective substances as a factor in freeze-thaw survival. Cryobiology 1, 298-300 (1965). [0441] 26. J. E. LOVELOCK, M. W. BISHOP, Prevention of freezing damage to living cells by dimethyl sulphoxide. Nature 183, 1394-1395 (1959). [0442] 27. M. R. Elliott, K. S. Ravichandran, Clearance of apoptotic cells: implications in health and disease. J Cell Biol 189, 1059-1070 (2010). [0443] 28. M. Lucas et al., Requirements for apoptotic cell contact in regulation of macrophage responses. J Immunol 177, 4047-4054 (2006). [0444] 29. J. Savill, I. Dransfield, C. Gregory, C. Haslett, A blast from the past: clearance of apoptotic cells regulates immune responses. Nat Rev Immunol 2, 965-975 (2002). [0445] 30. C. N. Serhan, J. Savill, Resolution of inflammation: the beginning programs the end. Nat Immunol 6, 1191-1197 (2005). [0446] 31. A. Mantovani et al., The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol 25, 677-686 (2004). [0447] 32. D. M. Mosser, J. P. Edwards, Exploring the full spectrum of macrophage activation. Nat Rev Immunol 8, 958-969 (2008). [0448] 33. S. Gordon, A. Pluddennann, Tissue macrophages: heterogeneity and functions. BMC Biol 15, 53 (2017). [0449] 34. S. Gordon, A. Pluddennann, The Mononuclear Phagocytic System. Generation of Diversity. Front Immunol 10, 1893 (2019). [0450] 35. W. J. de Villiers, I. P. Fraser, S. Gordon, Cytokine and growth factor regulation of macrophage scavenger receptor expression and function. Immunol Lett 43, 73-79 (1994). [0451] 36. G. Casella et al., IL4 induces IL6-producing M2 macrophages associated to inhibition of neuroinflammation in vitroand in vivo. J Neuroinflammation 13, 139 (2016). [0452] 37. L. Bosurgi et al., Transplanted mesoangioblasts require macrophage IL-10 for survival in a mouse model of muscle injury. J Immunol 188, 6267-6277 (2012). [0453] 38. L. Bosurgi et al., Vessel-associated myogenic precursors control macrophage activation and clearance of apoptotic cells. Clin Exp Immunol 179, 62-67 (2015). [0454] 39. L. Bosurgi et al., Macrophage function in tissue repair and remodeling requires IL-4 or IL-13 with apoptotic cells. Science 356, 1072-1076 (2017). [0455] 40. I. Montfort, R. Perez-Tamayo, Collagenase in experimental carbon tetrachloride cirrhosis of the liver. Am J Pathol 92, 411-420 (1978). [0456] 41. R. Perez Tamayo, Is cirrhosis of the liver experimentally produced by CCI4 and adequate model of human cirrhosis? Hepatology 3, 112-120 (1983). [0457] 42. J. Shi, K. Aisaki, Y. Ikawa, K. Wake, Evidence of hepatocyte apoptosis in rat liver after the administration of carbon tetrachloride. Am J Pathol 153, 515-525 (1998).

EQUIVALENTS

[0458] 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

[0459] 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.