Mini Experiment 4:
Seeding Titration Experiment on Transwell Filters and Porosity Test
What is the best amount of cells for the least porosity?
Use the area of each cell: use confluency and count of cells from cell culture notes to calculate. Need area of plate
Corning Costar Transwell filters + plates:
0.4 μm Pore Size; 12 mm Diameter; 1.0 cm2 Growth Area polycarbonate
Liposome size: 80nm ~ 150 nm
- Pour (find volume) media onto the Transwell filter
- Seed 80%, 85%, 90%, 95% confluent b.END3 cells into different wells
- Once the cells have adhered to the filter (form a barrier), add plain liposomes onto the filter
- Stain the bottom of the well to see how much liposomes were able to pass through barrier (?)
- The optimal confluency for b.END3 cells will be the percentage that allowed the least amount of liposome to pass through the barrier
Use polyester transwell?
- Are your cells growing well on non-porous surfaces (flasks) to begin with?
- Are you waiting until they are confluent in flasks before passaging them to inserts?
- Are you waiting too long after they are confluent before passaging them to inserts?
- Are you over-trypsinizing them?
- Are you using too high a passage (there are reports that BBB characteristics start to degrade past ~P30)?
This lab is using polycarbonate transwell filters:
We are coating our transwell inserts with collagen G (from Biochrom Cat. Nr. L-7213; dilution in PBS with 1% PS) at a conc. of 120µg/ml (!!) for about 30 min in the incubator before seeding out the cells. This conc. is dubble as high as the normal conc. for monolayers. The cell density we are using for seeding out is 1:1 for 6-well TW and 1:2 for 12-well TW.
In terms of Exp. B2:
- Growing b.END3 cells on transwell filter to simulate BBB
- Placing liposomes + EGF / negative control onto the filter and see how many can pass through the filter and end up in the well below
Check seeding for high confluencies and seeing which one will be the optimal amount for b.END3 cell adherence onto the transwell filter so that liposomes cannot pass through the filter without being in contact with b.END3
- Do we need to figure out the optimal concentration of liposomes (EGF vs. Non) too?
- Do cells continue to grow once seeded on filter? How long should we leave it for before adding liposomes?
Resources to answer questions:
|Confluency Multiplier||Cells||Days of incubation||Amount of Cells added||Amount of media added|
Adding media to cells: added towards inside of transwell filters first, then at midway added it towards the side of the well, then added cells right in the middle.
|0.9 2-day incubation|
|1.1 1-day incubation|
|1 2-day incubation||1.1 2-day incubation||1.2 2-day incubations|
For the purpose of this experiment, we will incubate with liposomes for 1 hour, to ensure liposomes move down through the liquid and through the pores if they are present. Protocol will be as follows:
Preparation of liposomes:
- Add 8.5µL of liposome-EGF to 2491µL media
- Prepare filter sterilizer (take out plunger, put syringe filter on, put over 15 mL tube) and put liposome with media through filter (by adding plunger)
Addition of Liposomes to wells:
- Take out media inside wells (2 each time) using P1000 very slowly, DO NOT TOUCH bottom of transwell filter
- Add 250µL media to the well (into the side)
- Add 250 µL liposomes into well
- Incubate for one hour
- Label microcentrifuge tubes
- Take media from inside of transwell filter and put into microcentrifuge tube
- Take out transwell filter using tweezers and take media from the bottom and put into a seperate microcentrifuge tube using P1000, do all these steps slowly
Changed conditions, so that for the ones seeding density is being titrated, cell incubation on transwell filters is two days instead of 1.
The wells at the bottom and the one away on the right top had much less volume of media inside them compared with the one in the top left. The one in the top left was also surrounded with wells that only had media inside them. The cause of lower volume is evaporation for sure, for next run, all wells being experimented on must be surrounded by other sample wells or wells with media/ pbs inside them to increase humidity and therefore decrease evaporation. For now added 250 ul of media to each depleted well. Expecting results from this experiment to be largely affected by this and quality of cells, and no replicates.
Starting time of incubation: 5:09
Day 2: everything was normal instead of following:
- Touched bottom of filter in down left corner of plate
- Realized should take out media first, then media inside the filter, then add media to outside, then add liposomes
- Start of incubation time: 4:45
September 27, 2016
Present: Angel, Ina
Task: Seed bEND3 cells onto transwell filter membranes
- 600 µL of media was loaded into the 2 wells of the 24-well plate (as per Corning media suggestions)
- BD Falcon 0.4 µm PET membranes were placed into the 2 wells and were left to perch on the 24-well plate
- 100 µL of media was added into the inserts
- The plate was allowed to incubate at 37 C for 1 hour in order to prime the system, as per suggestion by Corning
- bEND3 cells (P14) were counted using a haemacytometer
- A density of 4.67x104 cells (well A1) and 4.67x105 cells (well A3) were seeded (seeding density is based on an optimized seeding density suggested by Corning. The seeding density seemed reasonable when confirmed with papers by Guanglei Li., et al)
- Cells were left to incubate and grow overnight
Purpose: The purpose of this task was to seed bEND3 cells and find a way to either measure TEER, cell permeability or to image cells via confocal microscope
- Cell permeability test using Sodium Fluorescein
- Image cells via confocal microscopy
- Involves fixing the cells, staining with antibody/nuclear stain
- Cutting out the membrane
- Placing on a slide and gluing a coverslip on top