Cell therapy with polarized macrophages for tissue regeneration

Abstract

The invention provides an in vitro method for inducing macrophage polarization to an M2 phenotype. The method comprises the in vitro exposure of macrophages to repeated series of hypoxia-reoxygenation. Activated M2 macrophages obtained by this method overexpress molecules important for tissue remodeling and amelioration of inflammation, thus they are useful as cell therapy for tissue regeneration. The invention also provides pharmaceutical compositions and kits comprising the M2 macrophages obtained by the method, as well as a device for inducing hypoxia and re-oxygenation conditions on isolated macrophages according to the method.

Claims

1. An in vitro method for obtaining macrophages polarized to an M2 phenotype that comprises: a. subjecting isolated macrophages to 4 series of hypoxia-reoxygenation, and b. recovering the macrophages obtained after step (a), wherein the macrophages recovered after step (b) overexpress NGAL and IL10.

2. The method according to claim 1, wherein each series of hypoxia-reoxygenation comprises between 2 and 5 minutes of hypoxia followed by at least 45 seconds of reoxygenation.

3. The method according to claim 1, which comprises an additional step (a), between the steps (a) and (b), comprising subjecting the macrophages obtained after step (a) to a final reoxygenation step.

4. The method according to claim 3, wherein the final reoxygenation step is performed during no more than 1 hour and 30 minutes.

5. The method according to claim 1, wherein the macrophages of step (a) are monocytes.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. NGAL gene expression (mRNA) normalize to GAPDH in macrophages in culture subjected to 1, 2, 3, 4 or 5 hypoxia-reoxygenation series or cultured under standard conditions (control). *P<0.005.

(2) FIG. 2. IL10 gene expression (mRNA) normalize to GAPDH in macrophages in culture subjected to 1, 2, 3, 4 or 5 hypoxia-reoxygenation series or cultured under standard conditions (control). *P<0.005. **P<0.001.

(3) FIG. 3A. Shows a diagram of the device for inducing hypoxia and re-oxygenation conditions on isolated macrophages.

(4) FIG. 3B. Shows a perspective view of the device for inducing hypoxia and re-oxygenation conditions on isolated macrophages.

(5) FIGS. 4A-D. Show different embodiments of the removable chamber of the device of FIGS. 3A-B.

(6) FIG. 5. Expression of fibronectin (mRNA) normalize to GAPDH in the muscle collected from mice. PREC: mice with injury treated with the in vivo hypoxia-reoxygentation protocol. PREC+Macs M2: mice with injury treated with the in vivo hypoxia-reoxygentation protocol+M2 macrophages.

(7) FIG. 6. Expression of connective tissue grow factor (CTGF) (mRNA) normalize to GAPDH in the muscle collected from mice. PREC: mice with injury treated with the in vivo hypoxia-reoxygentation protocol. PREC+Macs M2: mice with injury treated with the in vivo hypoxia-reoxygentation protocol+M2 macrophages.

(8) FIG. 7. Expression of IL-6 (mRNA) normalize to GAPDH in the muscle collected from mice. PREC: mice with injury treated with the in vivo hypoxia-reoxygentation protocol. PREC+Macs M2: mice with injury treated with the in vivo hypoxia-reoxygentation protocol+M2 macrophages.

(9) FIG. 8. Expression of TWIST1 (mRNA) normalize to GAPDH in the muscle collected from mice. PREC: mice with injury treated with the in vivo hypoxia-reoxygentation protocol. PREC+Macs M2: mice with injury treated with the in vivo hypoxia-reoxygentation protocol+M2 macrophages.

EXAMPLES

Example 1

Effects of Short Periods of Anoxia-Reoxygenation on the Polarization of Peritoneal Macrophages

(10) 1.1. Objective.

(11) The goal was to assess if the exposure of the macrophages to 1, 2, 3, 4 or 5 anoxia-reoxygenation series (3anoxia-45 reoxygenation) promotes the polarization of macrophages to an M2 phenotype, with an increase in NGAL and IL10 expression compared to the control subjected to standard incubation conditions.

(12) 1.2. Experimental Design.

(13) a. Control group under oxygen standard conditions (CO.sub.2 5% plus atmospheric air).

(14) Peritoneal macrophages were extracted and isolated from six mice and cultured during 24 h under standard conditions.

(15) b. Groups subjected to 1, 2, 3, 4 or 5 anoxia (nitrogen 95%; CO.sub.2 5%; O.sub.2 0%).-reoxygenation series plus 1 h and 30 min of reoxygenation.

(16) Peritoneal macrophages were extracted and isolated from six mice and cultured during 24 h under standard conditions. Then, macrophages were subjected to 1, 2, 3, 4 or 5 anoxia-reoxygenation series (3anoxia-45 reoxygenation). Finally, macrophages were subjected to 1 h and 30 min of reoxygenation.

(17) 1.3. Material and Methods.

(18) For the extraction of peritoneal macrophages, 2.5 ml thioglycolate were intraperitoneally injected in 6 mice.

(19) This process was performed 6 times, one for each mouse:

(20) 1. Peritoneal macrophages were extracted in 20 ml PBS.

(21) 2. Cells were resuspended in 1 ml RPMI medium 10% PBS 1% P/S (penicillin/streptomicin).

(22) 3. Cells were counted and 3.5 million per well of live cells were seeded (Table 1).

(23) TABLE-US-00001 TABLE 1 % live Million of l of cells Mice Million cells live cells per well Million/well 1 64 60 38.4 91.14583333 3.5 2 39 55 21.45 163.1701632 3.5 3 72 66 47.52 73.65319865 3.5 4 30 66 19.8 176.7676768 3.5 5 31 60 18.6 188.172043 3.5 6 33 60 19.8 176.7676768 3.5

(24) Cells were incubated under standard conditions during 24 h.

(25) Afterwards, our intermittent anoxia protocol was performed in a hypoxia chamber under the following conditions: nitrogen 95% and CO.sub.2 5%, 0% O.sub.2. Groups to be assessed were the following:

(26) 1. Control under oxygen standard conditions (CO.sub.2 5% plus atmospheric air). Peritoneal macrophages in a well containing 2 ml RPMI medium 10% FBS 1% P/S during 24 h.

(27) 2. 1 anoxia-reoxygenation serie. Peritoneal macrophages in a well containing 2 ml RPMI medium 10% FBS 1% P/S. 1 serie consisting of 3 anoxia/45 reoxygenation+1 h and 30 min of reoxygenation.

(28) 3. 2 anoxia-reoxygenation series. Peritoneal macrophages in a well containing 2 ml RPMI medium 10% FBS 1% P/S. 2 series consisting of 3 anoxia/45 reoxygenation+1 h and 30 min of reoxygenation.

(29) 4. 3 anoxia-reoxygenation series. Peritoneal macrophages in a well containing 2 ml RPMI medium 10% FBS 1% P/S. 3 series consisting of 3 anoxia/45 reoxygenation+1 h and 30 min of reoxygenation.

(30) 5. 4 anoxia-reoxygenation series. Peritoneal macrophages in a well containing 2 ml RPMI medium 10% FBS 1% P/S. 4 series consisting of 3 anoxia/45 reoxygenation+1 h and 30 min of reoxygenation.

(31) 6. 5 anoxia-reoxygenation series. Peritoneal macrophages in a well containing 2 ml RPMI medium 10% FBS 1% P/S. 5 series consisting of 3 anoxia/45 reoxygenation+1 h and 30 min reoxygenation.

(32) Once each protocol ended in each group, cells were incubated under standard conditions during 1 h and 30 min. After this time, cells were harvested and frozen in dry pellet for the subsequent RNA extraction protocol.

(33) RNA was extracted from the frozen samples and a reverse transcription to cDNA was carried out. Total RNA from cells was isolated using the RNeasy mini kit following the manufacturer's protocol (Qiagen, Barcelona, Spain). RNA concentrations were calculated from A.sub.260 determinations using a Nanodrop ND-1000 (NanoDrop Technologies, Wilmington, Del., USA). cDNA was synthesized by using the iScript cDNA synthesis Kit from Bio-Rad according to the manufacturer's recommendations. Quantitative RT-PCRs were performed on a Bio-Rad iCycler iQ Real-Time-PCR detection system using SYBR Green RT-PCR detection Kit (Bio-Rad, Madrid, Spain) according to the manufacturer's instructions. Real-time PCR results were quantified using Gene Expression Macro (version 1.1) from Bio-Rad, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal control for stable expression (housekeeping gene).

(34) Several RT-PCRs were run in order to assess the NGAL and IL10 expression in each group.

(35) 1.4. Results.

(36) Although 1, 2, and 3 series of 3 anoxia-45 reoxygenation also lead to an increased NGAL expression, significance in the both parameters measured (NGAL and IL-10) only was achieved with 4 series, whereas 5 series decreased NGAL levels (FIGS. 1 and 2).

Example 2

Description of a Preferred Embodiment of the Device

(37) In FIG. 3A it can be appreciated a diagram of the proposed device for inducing hypoxia and re-oxygenation conditions on isolated macrophages according to the method previously described. In FIG. 3B there is a perspective view of said device.

(38) As can be seen in said figure, the essential elements of the device are a removable chamber (1) configured to house the isolated macrophages, a first gas conduction circuit (2) and a second gas conduction circuit (3).

(39) The first gas conduction circuit (2) comprises a first removable connection (4), which is preferably a stopcock, to the removable chamber (1) and a first gas source (5) and a second gas source (6). The first and second gas sources (5, 6) are connected to an electrovalve (7) which is configured to select the gas source from which the gas passes to the removable chamber (1).

(40) The first gas conduction circuit (2) can also comprise a press sensor (16) placed between the electrovalve (7) and the first removable connection (4). In other embodiments of the invention, the press sensor (16) is placed between the electrovalve (7) and the gas sensor (11) or safety valve (10) or HEPA filter (9).

(41) The second gas conduction circuit (3) comprises a second removable connection (8) to the removable chamber (1) and a connection to the external environment (15) or a connection to a vacuum device. The second gas conduction circuit (3) is configured to control the removal of a gas inside the interior of the removable chamber (1).

(42) The first gas conduction circuit (2) further comprises at least one HEPA filter (9) and/or safety valve (10) between the first gas source (5) and the valve (7) or between the second gas source (6) and the valve (7). Preferably, when the device comprises both elements, the safety valve (10) is placed between the first gas source (5) or the second gas source (6) and the valve (7).

(43) In this case, the safety valve (10) is meant to protect the first gas conduction circuit (2) against over pressures. That is the reason why it has to be placed in the inlets and outlets of the first gas conduction circuit (2).

(44) The device can also comprise, in the first gas conduction circuit (2), a gas sensor (11) placed between the valve (9) and the first removable connection (4) to the removable chamber (1). Since there is no possible to introduce gas sensors in the interior of the removable chamber (1), in order to assure the quantity of gas to be introduced, in a preferable embodiment of the invention there is a gas sensor (11) in the inlet/outlet of the gas from said removable chamber (1).

(45) Preferably, the first gas conduction circuit (2) further comprises a safety valve (10) and/or an HEPA filter (9) between the gas sensor and the first removable connection (4).

(46) The first removable connection (4) is preferably a luer-lock connector or a luer-lock valve.

(47) Additionally, the second gas conduction circuit (3) can comprise at least an HEPA filter (9) and/or a safety valve (10) between the second removable connection (8) and the a connection to the external environment (15) or the connection to a vacuum device. In cases in which both elements are present, the HEPA filter (9) is placed, preferably, between the second removable connection (8) and the safety valve (10).

(48) The second removable connection (8) is preferably a luer-lock connector or a luer-lock valve.

(49) In the second conduction circuit (3), the safety valve (10) is meant to protect said gas conduction circuit (3) against over pressures.

(50) The first removable connection (4) and the second removable connection (8) are configured to be connected to at least a reception connection (14) of the removable chamber (1). The at least one reception connection (14) is, in an embodiment of the invention, a stop lock with a luer-lock valve.

(51) The second gas conduction circuit (3) can also comprise a gas sensor (11) between the second removable connection (8) and the connection to the external environment (15) or the connection to a vacuum device.

(52) Also the second gas conduction circuit (3) can comprise at least an electrovalve and/or a pump (12) between the second removable connection (8) and the connection to a vacuum device or the connection to the external environment (15).

(53) Preferably, the removable chamber (1) is a centrifuge tube. In FIGS. 4a-d some different embodiments of the removable chamber (1) are shown. The device can also comprise a controller configured for receiving signals from the gas sensors (11) and, in accordance with the received signals, control the valve (7) and the electrovalve or pump (12) in order to select from which gas source (5, 6) fill the removable chamber (1).

(54) As previously described, the removable chamber (1) is preferably a tube and more preferably a centrifuge tube. It can comprise a plunger (13) which can be moved longitudinally through the chamber and said plunger (13) comprises, as seen in FIG. 4A, at least one port with one reception connection (14), preferably a luer-lock valve which can be coupled to a syringe or to any other suitable device to introduce or remove the cells from inside the removable chamber (1). Afterwards, the reception connection (14) can be hermetically coupled to the first and second gas conduction circuits (5, 6) to fill and remove gas from inside the removable chamber (1).

(55) In a particular embodiment (FIG. 4B), the plunger (13) comprises a common reception connection for inlet and outlet of a gas which can be connected to a syringe, a second reception connection (14b) that can be hermetically coupled to the first gas conduction circuit (2) and a third reception connection (14c) that can be hermetically coupled to the second gas conduction circuit (3).

(56) In another possible embodiment, as shown in FIG. 4C, the plunger (13) can be punched with a needle to introduce or remove the cells from inside the removable chamber (1) or to fill and remove a gas composition inside the removable chamber (1).

(57) In another embodiment (FIG. 4D), the plunger (13) can be punched with a needle (14a) to introduce or remove the cells from inside the removable chamber (1) and comprises a first reception connection (14b) that can be hermetically coupled to the first gas conduction circuit (2) and a second reception connection (14c) that can be hermetically coupled to the second gas conduction circuit (3).

(58) A description is made of an example process carried out in the device. In this case, a blood sample from an individual is introduced, using a syringe, in a tube (the removable chamber (1)) and submitted to centrifugation in order to obtain a plurality of cell fractions. Afterwards, a second syringe is connected to the reception connection (14) (luer-lock) of the removable chamber (1) and, moving the plunger (13) downwards through the removable chamber (1), the syringe is filled with an isolated cell fraction. This process can be repeated several times until the cell fraction of interest is separated in an individual syringe.

(59) The cell fraction of interest is introduced in a removable chamber (1) of the device. This removable chamber (1) is connected to the device and is first filled with a first gas composition (N.sub.2) displacing the previous gas composition inside the removable chamber (1) through the second gas conduction circuit (3), and later filled with a second gas composition (synthetic air) displacing the first gas composition inside the removable chamber (1) through the second gas conduction circuit (3). This process is repeated four times.

(60) The removable chamber (1) is disconnected from the device, a syringe is connected to the reception connection (14) and, moving the plunger (13) downwards through the removable chamber (1), the cell fraction is recovered in the syringe.

Example 3

In Vivo Therapy for Treating Injured Tissue

(61) 3.1. Experimental Design and Procedure.

(62) a. Mice without injury (Control)

(63) b. Mice with an injury not treated

(64) c. Mice with an injury treated with an in vivo ischemia reperfusion protocol (PREC)

(65) d. Mice with an injury treated with an in vivo ischemia reperfusion protocol (PREC)+M2 macrophages

(66) An injury in the mice leg muscle was performed by laceration using a 5 mm diameter biopsy.

(67) After 48 h

(68) Peritoneal macrophages were extracted and isolated from mice (d) and polarized to an M2 phenotype using the procedure described in example 1.

(69) The legs of mice (c) and (d) were surrounded with a plastic track above the twin. Once it is well caught, it begins to tighten to make it a restriction of the flow during 3 minutes. After this period the track is released and the blood flow was recovered during 45 seconds. Afterwards the same ischemia reperfusion process was repeated 2 times.

(70) After the PREC protocol, M2 macrophages were injected to mice (d).

(71) After 96 hours

(72) The animals were sacrificed. Once the muscle was collected, it was cut in two parts and inserted in dry ice. Subsequently, the tissue was frozen at 80 and the RNA was extracted from a single piece using trizol. Due to the high values of RNA obtained in tissue, it was diluted in half with RNAse free water, the CDNA was made and the expression of the following genes was quantified:

(73) FIBRONECTIN (FIG. 5)

(74) CTGF (FIG. 6)

(75) IL-6 (FIG. 7)

(76) TWIST 1 (FIG. 8)

(77) The fibronectin, connective tissue grow factor (CTGF) and IL-6 markers were significantly reduced in the groups of animals receiving the treatment, indicating that the therapy is capable of reducing markers of fibrosis and inflammation. The increase of Twist1 marker also indicates that the treatment favors cell proliferation. The in vivo therapy described herein is therefore useful in the amelioration of inflammation and scar formation and for tissue remodeling.