170
- Cell culture plates: 6-well plates, 96-well ultralow attachment
plates. - Cell culture media: RPMI1640 (see Subheading 2.1).
- Tumor spheres formation medium: RPMI1640, 50× B27,
20 ng/mL epidermal growth factor (EGF), 10 ng/mL basic
fibroblast growth factor (FGF), 5 μg/mL insulin. Add 10 μg
EGF, 5 μg FGF, 2.5 μg insulin, and 0.5 mL B27 supplement
to 500 mL of RPMI1640 media (see Note 8). Store at 4 °C. - Cell culture medium: RPMI1640 (see Subheading 2.1).
- Cancer cells: SP-positive, SP-negative.
- Animal: Non-obese diabetic/severe combined immunodefi-
ciency (NOD/SCID) (see Note 9). - Anesthetic agents: 1–3% isoflurane, 80–100 mg/kg ketamine
(intraperitoneal) (see Note 10). - Syringe: 1 mL with 27- or 30-gauge needle.
- Digital caliper.
3 Methods
Unless otherwise mentioned, perform all the experiments at room
temperature.
- Take the cryovial containing esophageal adenocarcinoma cells
(OE33) from liquid nitrogen facility and transfer them into a
37 °C water bath. - Quickly thaw the cells by gently swirling the vial in water bath
at 37 °C (see Note 11). - Transfer the vial into a biosafety cabinet and wipe out site of
the vial with 80% ethanol. - Add pre-warmed complete growth media (2 mL) in a 15 mL
centrifuge tube and add the thaw cells dropwise into the tube. - Centrifuge the cell suspension at approximately 400 g for
3–5 min. - Check the cell pellet after centrifugation and aseptically decant
the supernatant without disrupting the cells pellet. - Add 2 mL PBS and resuspend the cells pellet with slow pipetting.
- Centrifuge the suspension at 400 × g for 3–5 min and discard
the supernatant. - Resuspend the cell pellets in complete growth media (contain-
ing FBS) and transfer them into an appropriate cell culture
flask containing recommended growth media and incubate in
the CO 2 incubator at appropriate conditions (see Note 12).
2.3 In Vitro
Functional Assay
2.4 In Vivo
Transplantation
3.1 Cell Culture
Farhadul Islam et al.