MSC/Macrophage co-cultures

MATERIALS

1.   Lymphoprep density gradient medium: LYS 3773; Axis shield

2.   KO-DMEM media: 10829-018; Gibco

3.   RPMI 1640 media: 11875-093; Gibco

4.   GlutaMAX (100X) : 35050-061; Gibco

5.   Sodium Pyruvate (100X): 11360-70; Gibco 

6.   Penicillin – Streptomycin (100X): 15140-122; Gibco

7.   Australian origin Foetal bovine serum (FBS): RM9951; Himedia

8.   M-CSF: 574808; BioLegend

9.   Lipopolysaccharide (LPS) from E. coli: L2880; Sigma Aldrich

10.  Human IFNg: I 3265; Sigma Aldrich

11.  Human IL-4: GF429; Merck

12.  12 well Multi well plates: 353053; Falcon

13.  Cell culture insert (12 well, 0.4 µm): 353180; Falcon

14.   PMA (Phorbol 12-myristate 13 acetate): P1585; Sigma Aldrich

15.   Dulbecco's phosphate-buffered saline (DPBS): 14190-144; Gibco

16.   0.25% Trypsin-EDTA: 25200-072; Gibco

17.   T-25 culture flask: 353108; Falcon 

EQUIPMENT 

1.   Benchtop Centrifuge – Eppendorf 5810R (Thermo Fisher Scientific)

2.   Hemacytometer - Rohem

3.   Humidified 5% CO₂ incubator – Hera Cell 240; Thermo Electron Corporation

4.   Inverted Microscope - Nikon Eclipse TE2000-S

5.   Pipettes – Eppendorf Research plus

6.   Bio safety cabinet – Thermo Scientific MSC advantage (Class II microbiological safety cabinet)

RECIPES 

1.  BM-MSC culture media (regular culture and expansion): KO-DMEM + 10% FBS + 1X GlutaMAX.

2.  BM-MSC culture media (for plating in inserts and co-culture): RPMI 1640 + 10% FBS + 1X GlutaMAX + 1X sodium pyruvate.

3.  THP-1 derived M_o macrophage polarization media: RPMI 1640 + 10% FBS + 1X GlutaMAX + 1X sodium pyruvate + Phorbol Myristate Acetate (PMA), 20 nM.

4.  THP-1 derived M1 macrophage polarization media: RPMI 1640 + 10% FBS + 1X GlutaMAX + 1X sodium pyruvate + LPS-100 ng/ml + IFNg-15 ng/ml.

5.  THP-1 derived M2 macrophage polarization media: RPMI 1640 + 10% FBS + 1X GlutaMAX + 1X sodium pyruvate + IL-4 – 10 ng/ml.

6.  Mo primary macrophage culture media: RPMI 1640 + 10% FBS + 1X GlutaMAX + 1X sodium pyruvate + M-CSF (50 ng/ml).

7.  M1 primary macrophage polarization media: RPMI 1640 + 10% FBS + 1X GlutaMAX + 1X sodium pyruvate + LPS-50 ng/ml + IFNg- 10 ng/ml.

8.  M2 primary macrophage polarization media: RPMI 1640 + 10% FBS + 1X GlutaMAX + 1X sodium pyruvate + IL-4 -20 ng/ml.

Note: To all media add 1x Penicillin-Streptomycin if required 

PROCEDURE

All the procedures are performed under sterile conditions (Class-II Biosafety hood).

ESTABLISHING MACROPHAGE CULTURE AND DIFFERENTIATION STATES

THP-1 MONOCYTE CELL LINE DERIVED MACROPHAGE CULTURE AND POLARIZATION PROTOCOL

1. To differentiate the suspended culture of THP-1 monocytes to naïve macrophages (Mo), THP-1 cells are seeded at a density of 75 x 10³ cells/cm² in complete RPMI media supplemented with 20 nM PMA (recipe 3) and placed at 37°C in a humidified 5% CO₂ incubator for 48 hrs.

2.  For M1 polarization, remove the PMA containing media and add complete RPMI 1640 media supplemented with 100 ng/ml of LPS and 15 ng/ml of IFNg (recipe 4) to the semi adherent Mo macrophage and incubate for another 48 hrs.

3. For M2 polarization, THP-1 cells are cultured in RPMI 1640 complete media containing 20 nM PMA for a duration of 6 hrs after which they are induced to M2 state by adding 10 ng/ml of IL-4 (recipe 5) in the presence of PMA and cultured for another 18 hrs.

  BLOOD MONOCYTE DERIVED PRIMARY MACROPHAGE CULTURE AND POLARIZATION PROTOCOL 

1.  Blood was mixed with equal volume of basal RPMI 1640 media and carefully layered over Lymphoprep (half the total volume of diluted blood) and centrifuged at 1800 rpm for 25 mins The brakes of the centrifuge were turned off during this step to prevent disintegration of the buffy coat layer. 

2.  After the spin, the top layer (pale yellow in colour) containing platelets was carefully removed by a pipette to minimize contamination of buffy coat with platelets.

3.   The buffy coat containing PBMCs was carefully collected and transferred to a 15 ml falcon after which DPBS was added to it and centrifuged at 1200 rpm for 8 mins to wash the PBMCs. This step was repeated thrice.

4.  After the final wash, DPBS was removed, and the base of the falcon was gently tapped to loosen the pellet. 1 ml of complete RPMI 1640 media containing 50 ng/ml of M-CSF (recipe 6) was added to the pellet and then resuspended with a pipette a couple of times to get a single cell suspension.

5. A cell count was taken and 2 x 10⁶ PBMCs per well of a 12 well plate was plated in 1 ml of complete RPMI 1640 media containing M-CSF (50 ng/ml) (recipe 6). The plate was then kept at 37°C in a humidified 5% CO₂ incubator and left undisturbed to facilitate attachment of monocytes and their subsequent differentiation to Mo macrophage.

6.  At day 3.5, media along with the unattached cells was removed and fresh complete RPMI media containing 50 ng/ml of M-CSF (recipe 6) was added. The plate was then placed in a 5% CO₂ incubator and the cells were cultured for another 3.5 days. 

7.  On the afternoon of day 7, Mo macrophages thus obtained were polarized to M1 and M2 macrophages. For M1 polarization, complete RPMI 1640 media supplemented with 50 ng/ml LPS + 10 ng/ml IFNg (recipe 7) was used and for M2 polarization complete RPMI media supplemented with 20 ng/ml IL-4 (recipe 8) was used. The cells were cultured in the presence of M1 and M2 polarization media for 48 hrs to obtain the desired M1 and M2 phenotype.

MACROPHAGE/MSC CO-CULTURE PROTOCOL 

THP-1 DERIVED MACROPHAGE /MSC CO-CULTURE

1. 6- 12 hrs prior to initiation of M1 polarization, primary human bone marrow derived MSCs from a T-25 flask are trypsinized and plated in a 12 well 0.4 µm cell culture insert in complete RPMI 1640 media (recipe 2) at a seeding density of 30 x 10³ cells/insert to maintain a 1:10 ratio of MSC to macrophages. Same volume of media is maintained in the well in which the insert is placed. The cells are cultured in the insert for overnight to facilitate attachment. On the next day, immediately after initiation of M1 polarization, the inserts containing MSCs are placed along with the macrophages to establish co-cultures. The co-cultures are terminated after 48 hrs.

2. Similarly, for M2/MSC co-culture, 6- 12 hrs prior to initiation of M2 polarization, MSCs from a T-25 flask are trypsinized and plated in 12 well, 0.4 µm cell culture insert in complete RPMI 1640 media (recipe 2) at a seeding density of 30 x 10³ cells/insert. One the next day, 6 hrs after PMA stimulation and immediately after exposure to M2 polarization stimuli, the inserts containing MSCs are placed along with the macrophages for another 18hrs after which the co-cultures were terminated. 

BLOOD MONOCYTE DERIVED PRIMARY MACROPHAGE /MSC CO-CULTURE

1. 6- 12 hrs before macrophage polarization initiation, MSCs being cultured in a T-25 flask are trypsinized and plated in a 12 well, 0.4 µm cell culture insert at a seeding density of 50 x 10³ cells/insert. The MSCs were seeded in complete RPMI 1640 media (recipe 2) and the volume of media in the insert and in the well in which the insert is placed was maintained at 1 ml. The cells were then cultured overnight at 37°C in a humidified 5% CO₂ incubator to facilitate their attachment as well as enable their acclimatization to RPMI 1640 media.

2. After overnight culture, inserts containing MSCs are transferred (18^th hour) to plates containing M1/M2 polarized macrophages and placed along with them to establish and initiate the co-culture in complete RPMI 1640 media (recipe 2). The plates are then placed at 37°C in a humidified 5% CO₂ incubator and MSC-macrophage co-cultures were continued for another 30 hrs after which they were terminated.