TITLE: Human Brain Vascular Pericytes (HBVP) Culture and Plating
AUTHORS: Rayan Khaddaj, Maxime Bernad, Daniel Manrique-Castano

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol implements the culture of Human Brain Vascular Pericytes (HBVP) and is suitable for several research techniques, including immunohistochemistry, flow cytometry, and Protein or RNA analysis.</div></div>

[STEPS]
?. [Cell Culture]
Prepare and warm  of Pericyte Complete Medium (PCM, see materials section) at   for .
50 mL
37 °C
?. [Cell Culture]
Retrieve form   the Human Brain Vascular Pericytes (HBVP) and place them at  for .
-130 °C
37 °C
As frozen HBVP contain DMSO, cell exposure to this reagent at    should be minimal. Therefore, incubate only as long as necessary until the aliquot is in liquid form.
?. [Cell Culture]
Place  (~500.000 cells) of HBVP in a 75cm² Cell Culture Flask filled with  of PCM.
1 mL
9 mL
Cell culture can also be performed in smaller Cell Culture Flasks if fast confluence or reduced cell number is required. In those cases, adjust the PCM volume (i.e   for 25cm² Cell Culture Flask).
?. [Cell Culture]
Perform gently crosswise movements to spread the cells over the container's surface, and visually check cell dispersion under a microscope.
?. [Cell Culture]
Incubate the cells at  until reaching a confluence of 70-80% before proceeding with cell plating.
37 °C
In a 75cm² Cell Culture Flask it is expected that 70-80% confluence results in 6-7 million cells.
When HBVP are taken from -130 frozen aliquots, the PCM must be replaced daily during the first two days to eliminate any traces of DMSO (see step 20). After, replace the medium every two days until the desired confluence is reached.
?. [Glass Coverslip Preparation]
The steps contained in this section (Glass Coverslip Preparation) are required if the experimental approach entails immunohistochemistry. Otherwise, If mounting in coverslips is not necessary (i.e. protein extraction), the researcher can proceed to cell detachment.
Here, the Glass Coverslip coating was performed with Matrigel, for specific purposes. In most scenarios, Poly-D-Lysine coated coverslips are used. To use Poly-D-Lysine, prepare in a sterile environment  /  (Mili-Q water) of    and filter it using a Syringe filter. Cover the Glass Coverslip with o  or   of the diluted Poly-D-Lysine and incubate at   for  . Subsequently, wash with sterile DPBS. At this point, coverslips are ready for cell plating.
?. [Glass Coverslip Preparation]
Slowly thaw aliquoted Matrigel by transferring it from  to a recipient with minced ice.
-20 °C
?. [Glass Coverslip Preparation]
Dilute 1:30 the Matrigel in cooled DPBS according to the required volume per wheel ( for 12 wheels plate, for 6 wheels plate ) and filter the solution using a sterile Syringe and a   Syringe filter.
on ice
200 µl
400 mL
2 µl
?. [Glass Coverslip Preparation]
Place cold (stored at ) Glass Coverslips in each wheel and add  70% ethanol for  with subsequent suction.
-20 °C
1 mL
?. [Glass Coverslip Preparation]
, add  for 12 wheels plate or for 6 wheels plate of filtered matrigel and incubate the plate at   for  .
on ice
200 µl
400 mL
37 °C
?. [Glass Coverslip Preparation]
When matrigel incubation is finished, wash the wheels three times with PCM ( for 12 wheels plate, for 6 wheels plate) and subsequently, aspirate the media.
200 µl
500 mL
?. [Cell Detachment]
Before starting the procedure, make sure that PCM, DPBS, and Trypsin-EDTA solution are warmed at  for
37 °C
?. [Cell Detachment]
When cells have reached 70-80% confluence (6-7 million cells), take them from the incubator, carefully suction the PCM, and wash 1 time with DPBS.
?. [Cell Detachment]
After suctioning DPBS, add  of trypsin-EDTA solution and incubate at  for  to achieve cell detachment from the flask.
3 mL
37 °C
If culture is performed in a smaller Cell Culture Flask, adjust the trypsin-EDTA volume accordingly. For 25cm² Cell Culture Flask,   of the solution is enough.
?. [Cell Detachment]
Check under the microscope that cell detachment was achieved, and add  of PCM (three times the trypsin-EDTA volume) to stop the chemical reaction.
9 mL
If culture is performed in a smaller Cell Culture Flask, adjust the PCM volume to be three times the trypsin-EDTA volume.
?. [Cell Detachment]
Repeatedly pipette the medium contained in the Cell Culture Flask to ensure complete detachment of cells that may still be adhered to the surface.
?. [Cell Detachment]
Suction the whole culture media containing the detached cells, and place it in a  sterile Falcon tube.
50 mL
?. [Cell Detachment]
To separate the cells from the PCM and remaining trypsin-EDTA solution, centrifugate at
Centrifuge: 1000 33, 5 min
?. [Cell Detachment]
When centrifugation is finished and cells are attached to the bottom of the Falcon tube, carefully remove the supernatant and add PCM with gently pipetting to resuspend the cells (check cases).
Known cell numberThis protocol estimates that under a confluence of 70-80% in a 75cm2 Cell Culture Flask, the researcher can retrieve a total of 7 million cells. In this scenario, cells are resuspended in 7 ml PCM to obtain 1 million cells/ml.Cell countingIf required or desired, the researcher can conduct at this point manual or automatic cell counting according to equipment availability. This is usually done by 1:1 incubation of 10 ul of Trypan Blue and a 1/10 diluted cell sample. Then, resuspension in PCM is performed according to the desired cell concentration per milliliter.
?. [Cell Detachment]
At this point, cells are ready to be plated (See next section) or frozen. If the researcher wishes to freeze the cells, they must be resuspended in PCM without FBS and add 20% v/v Dimethyl Sulfoxide (DMSO).
?. [Cell Detachment]
To freeze the cells, add  (1 million cells) of cell suspension in  Eppendorf tubes, and place them in a recipient filled with isopentane. Transfer the recipient to a   refrigerator for  , and finally place them at  for further use.
1 mL
1.5 mL
-80 °C
-130 °C
During the freezing and thaw cycles, a loss of ~50% of cells is expected. Therefore, it is recommended to freeze 1 million cells, in order to seed at least 500.000 cells in the next passage.  (see steps 2-3).
?. [Cell Plating]
Add  (for 6 wheels plate) or   (for 12 wheel plates) of PCM to each wheel containing the Matrigel Coated Glass Coverslips (see Glass Coverslip Preparation section), followed by  (for 6 wheels plate) or   (for 12 whell plate) of PCM-suspended pericytes.
1 mL
750 mL
0.5 mL
250 mL
?. [Cell Plating]
Incubate the wheel plate at  until the desired confluence is achieved.
37 °C
70-80% Confluency in 6 or 12 wheels plates is achieved in 1-3 days depending on the plated cell density.