Procedures of MDCK2 cyst formation

Aim

Procedures of MDCK2 cyst formation

Materials

MDCK2 cells (7.5 × 10³ cells in 150 µL medium per well )

Chamber Cover 8 well (Matsunami, SCC-008)

1.5 mL tube × 2

5.0 mL tube × 2

DMEM (low glucose)/10% FBS (hereafter simply "DMEM")

Matrigel Growth Factor Reduced Basement Membrane Matrix (Corning, 354230)

Methods

Drop 40 µL of the Matrigel solution into the center of each well of the Chamber Cover, and use the tip of the pipette to evenly spread the solution across the well.

Although a meniscus form at the edge of the well, ensure that the center is spread flat.

After the required number of wells is coated, place the Chamber Cover in 37°C incubator and allow the Matrigel to solidify for at least 15 minutes.

Solidification for up to 40 minutes has also been confirmed to be acceptable.

Prepare a carefully pipetted cell suspension at the desired concentration.

Prepare an equal volume of DMEM/4% Matrigel (e.g., 1.2 mL for 8 wells).

Mix the cell suspension and DMEM/4% Matrigel in a 5.0 mL tube to obtain DMEM/2% Matrigel containing 7.5 × 10³ cells per well.

Carefully add 300 µL of the cell–Matrigel mixture into each Matrigel-coated well.

Incubate the Chamber Cover in a 37°C incubator.

Every 2 days, carefully aspirate approximately 100 µL of the medium from the top of each well using a pipetman, and gently add 300 µL of fresh DMEM/2% Matrigel to perform a medium change.

チャンバーカバーのwellの中心に40 µL/wellのマトリゲルを滴下し、滴下したチップの先を使ってwell内に均等に塗り広げる

wellの端はメニスカスが生じるものの、中心付近はフラットに塗り広げる

必要な数のwellに塗り広げたら、チャンバーカバーを37℃のインキュベータに入れ、少なくとも15分固化させる

40分でも問題がないことを確認済み

入念にピペッティングした、目的の濃度の細胞懸濁液を用意する

細胞懸濁液と同量のDMEM/4% Matrigelを用意する (8 wellの場合は1.2 mL)

5.0 mLチューブに細胞懸濁液とDMEM/4% Matrigelを加え、1 wellあたり7.5 × 10³ cellsを含むDMEM/2% Matrigelに調製する

Matrigelを塗り広げたチャンバーカバーをインキュベータから取り出し、300 µL/wellの混合溶液を慎重に滴下する

チャンバーカバーをインキュベータに入れて培養する

2日おきにピペットマンで慎重に100 µL/well程度の上層をアスピレートし、300 µL/wellのDMEM/2% Matrigelを慎重に加え、培地交換する

Remarks

MDCK2 cysts form faster than MCF10A cysts.

By day 2, they typically reach a diameter of ~30 µm, and a lumen becomes visible.

MDCK2のCystはMCF10AのCystと比較して速く形成される

2日後には直径が約30 µmになりlumenが観察されるようになる