Recovery and Expansion of Patient-Derived Glioblastoma Cells After Long-term Cryopreservation

Materials and reagents

Biological materials

1. Patient-derived glioblastoma cells (originally collected, isolated, and cryopreserved over a decade ago by a previous researcher [9]; subsequently thawed and utilized in our recently published study [10] and this protocol development); see Supplementary Materials of [10] for patient data details

Reagents

1. Dulbecco’s modified Eagle medium (DMEM), high glucose (Thermo Fisher Scientific, Gibco, catalog number: 12100046)

2. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F7524)

3. Penicillin-streptomycin mixture (double antibiotics, 100×) (Servicebio, catalog number: G4003)

4. Trypsin-EDTA (0.25%) (Thermo Fisher Scientific, Gibco, catalog number: 25200072)

5. Matrigel^® basement membrane matrix, LDEV-free, 10 mL (Corning, catalog number: 354234)

6. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S5886)

7. Sodium phosphate dibasic (Na₂HPO₄) (Sigma-Aldrich, catalog number: 106586)

8. Potassium phosphate monobasic (KH₂PO₄) (Sigma-Aldrich, catalog number: P0662)

9. APC anti-human CD44 antibody, dilution 1:100 (BioLegend, catalog number: 38806)

10. PE/Cyanine7 anti-human CD133 antibody, dilution 1:100 (BioLegend, catalog number: 393909)

11. Anti-nestin antibody, dilution 1:250 (Sigma-Aldrich, catalog number: MAB5326)

12. Anti-SOX2 antibody, dilution 1:250 (Sigma-Aldrich, catalog number: AB5603)

Solutions

1. Matrigel coating solution (see Recipes)

2. Optimized recovery medium (see Recipes)

3. Standard culture medium (see Recipes)

4. 1× phosphate-buffered saline (PBS) (see Recipes)

5. Trypsin solution 0.05% (see Recipes)

Recipes

1. Matrigel coating solution

*This recipe provides an example for a Matrigel stock concentration of 9.0 mg/mL. The volume of basal DMEM (high glucose) added to the Matrigel aliquot was adjusted according to the lot-specific concentration reported in the manufacturer’s certificate of analysis for each Matrigel lot. To ensure a standardized final concentration of 0.3 mg/mL, the required dilution volume was calculated for each lot using the following formula:

V_medium = [(C_stock × V_aliquot)/0.3 mg/mL] - V_aliquot

V_medium is the required dilution volume of basal DMEM (high glucose) medium;

C_stock is the lot-specific concentration of the Matrigel stock (such as 9.0 mg/mL);

V_aliquot is the volume of the frozen Matrigel aliquot (such as 0.2 mL);

Example calculation in this recipe:

V_medium = [(9 × 0.2)/0.3] - 0.2 = 5.8 mL

Matrigel basement membrane matrix stock was stored at -80 °C until use or aliquoting. Aliquot volumes were prepared according to the manufacturer’s recommended working concentration range for coating (0.2–0.3 mg/mL); in our laboratory, a concentration of 0.3 mg/mL in 6 mL was routinely used as the working solution. For aliquoting, the Matrigel stock was thawed on ice and dispensed into pre-chilled 15 mL centrifuge tubes, which were then stored at -20 °C until use. To prepare the Matrigel coating solution, an aliquot was diluted with ice-cold DMEM (high glucose) to prevent gel formation upon warming. To ensure homogeneity, the diluted solution was incubated overnight at 4 °C. Although fresh preparation was preferred, working solutions were stored at 4 °C for no longer than one week.

2. Optimized recovery medium

Fresh preparation is preferred, but if stored, the medium should be kept at 4 °C for no longer than one week to minimize the risk of contamination and degradation of nutrients. Warm the medium at 37 °C before use.

3. Standard culture medium

The medium should be kept at 4 °C for no longer than 2–4 weeks to minimize the risk of contamination and degradation of nutrients. Warm the medium at 37 °C before use.

4. 1× PBS

In our laboratory, 1,000 mL of 1× PBS was prepared by dissolving the chemical powders listed in the recipe in Milli-Q water. The solution was stirred until fully dissolved, and the pH was adjusted to approximately 7.4. The buffer was then brought to a final volume of 1,000 mL and sterilized by autoclaving. After sterilization, the solution was allowed to cool overnight before use. Sterile PBS was stored at room temperature for up to 6 months. Sterile 1× PBS from commercial sources can be used when preparation is not preferred.

5. Trypsin solution (0.05%)

Store 0.05% trypsin solution at 4 °C for no longer than 1–2 weeks for reliable enzyme activity. Warm at room temperature before use.

Laboratory supplies

1. 6-well clear TC-treated multiple well plates, individually wrapped, sterile (Corning, catalog number: 3516)

2. 25 cm² rectangular canted neck cell culture flask with vent cap (Corning, catalog number: 430639)

3. 75 cm² U-shaped canted neck cell culture flask with vent cap (Corning, catalog number: 431464U)

4. Centrifuge tubes with flat cap, 15 mL, sterilized (JET Biofil, catalog number: CFT011150)

5. Centrifuge tubes with flat cap, 50 mL, sterilized (JET Biofil, catalog number: CFT011500)

6. 2 mL internal threaded polypropylene cryogenic vial, self-standing with round bottom (Corning, catalog number: 430488)

7. 10 μL pipette tips, non-filtered, clear (Corning, Axygen^®, catalog number: T-300)

8. 1–200 μL pipette tips, graduated, yellow (Corning, catalog number: 4112)

9. 1,000 μL pipette tips (maximum working volume 1,250 μL), graduated, natural (Corning, catalog number: 4114)

Equipment

1. CO₂ incubator (Thermo Scientific, Forma^TM Steri-Cycle^TM Incubator, 184 L, Polished Stainless Steel, model: 371)

2. Class II biological safety cabinet (Thermo Fisher Scientific, model: MSC-Advantage BSC 1.8)

3. Inverted microscope (Nikon, model: Eclipse TS100)

4. Water bath at 37 °C (Julabo, model: SW23)

5. Pipetman P1000, 100–1,000 μL (Gilson)

6. Motorized pipette controller (Corning, model: Stripettor^TM Ultra Pipet Controller 4099)

7. Disposable serological pipette 5, 10, and 25 mL (Wuxi NEST Biotechnology)

8. Laboratory/medical refrigerator at 37 °C (Panasonic, model: MPR-1411)

9. Liquid nitrogen tank (Thermo Fisher Scientific, model: CY509109)

Procedure