Origin and Features of the KNS-89
The KNS-89 cells is derived from the brain tissue of a 75-year-old male patient with glioblastoma and is widely used in cancer research, particularly in studies on the fundamental mechanisms of glioma and drug screening. Specifically, the KNS-89 cell line originates from a brain tissue sample of a patient diagnosed with astrocytoma. KNS-89 cells exhibit an epithelial-like morphology and adherent growth properties, making them suitable for in vitro culture. These characteristics establish the KNS-89 cell line as an important model for studying the development and progression of glioblastoma as well as drug responses.
Applications of the KNS-89
Basic Research: Used to explore the pathogenesis and biological characteristics of glioma.
Drug Screening: Serves as a model system for in vitro screening of anti-tumor drugs and efficacy evaluation.
High-Throughput Screening: Can be applied in toxicological studies, such as assessing the effects of different compounds on cell proliferation.
Morphological Observation and Live Cell Station Detection of KNS-89 at Different Densities
Figure 1.KNS-89 cells cultured on Ucallm® Ultra-Low Attachment Surface forms tumor spheroids. KNS-89 cells were planted in 96-well ultra-low attachment plates at concentrations of 500, 1000, 2000, 4000, and 8000 cells per well. Live-cell imaging was conducted at 24, 48, 72, 96, and 120 hours after seeding. Scale bars represent 200 μm.
Method
Culture Conditions
KNS-89 cells: 89% DMEM(High Glucose)+10% FBS+1% P/S
Cell recovery
1) Remove the KNS-89 cells from liquid nitrogen and quickly place them in a 37℃ water bath. Gently shake the cryovial to thaw the freezing medium.
2) After thawing, transfer the cells to a centrifuge tube containing 3 mL of culture medium. Centrifuge to collect the cells: 1000 rpm for 5 minutes at room temperature. Discard the supernatant.
3) Resuspend the cell pellet in complete medium. Seed the cell suspension into a culture dish, gently pipet to mix, and culture in a standard incubator at 37℃, 21% O2, and 5% CO2.
Cell Passaging
1) When cell density reaches 80%, perform subculturing.
2) Discard the old culture medium and wash the cells once with PBS.
3) Add 1-2 mL of 0.25% trypsin to digest the cells. Incubate at 37℃ for 1-2 minutes. Observe under a microscope; digestion is complete when cells detach and become rounded.
4) Quickly discard the trypsin. Add complete medium and pipet to create a single-cell suspension. Passage at a ratio of 1:2 to 1:4. Transfer to a standard incubator at 37℃, 21% O2, and 5% CO2 for expanded culture.
Cell Seeding
Harvest KNS-89 cells in the logarithmic growth phase with good growth condition. Seed them into a 96-well U-bottom ultra-low attachment plate at densities of 500, 1000, 2000, 4000, and 8000 cells per well respectively (for one plate), with 5 replicate wells per density. Add 100 μL of sterile PBS to the peripheral wells of the plate. Place the 96-well U-bottom ultra-low attachment plate in the live cell station for cultivation and imaging.
*Note: The live cell station is installed inside a CO2 constant temperature incubator , pre-warmed for 30 minutes, and maintained at 37℃, 21% O2, 5% CO2, and saturated humidity.
Medium Change
Change the medium for KNS-89 cells every 24 hours (the plate in the live cell station is changed simultaneously. Note: The medium change frequency differs for the various density wells: the 1000 and 2000 cells/well densities have a lower change frequency compared to the 4000 and 8000 cells/well densities). Continue this for 120 hours, totaling 5 medium changes.
During medium change, carefully aspirate and discard the old medium, handling all 5 replicate wells of one density at once. Then quickly replenish with 100 μL/well of fresh complete medium.
Materials and Instruments
able 1 Main equipment
Name | Manufacturer | Catalog Number |
CO2 Incubator | Thermo | 3111 |
Live Cell Station | NanoEntek Inc | JuLI Stage |
Inverted Microscope | OLYMPUS | IX73 |
96-well Ultra-Low Attachment（U-bottom）Cell Culture Plate | Ucallm | L1096UA |
Table 2 Major Reagents
Name | Manufacturer | Catalog Number |
KNS-89 | JCRB(Japanese Collection of Research Bioresources ) | IFO50360 |
DMEM High Glucose Cell Culture Medium | Gibco | 11965092 |
FBS | Gibco | 10099141 |
Penicillin-Streptomycin Solution | Gibco | 15140122 |
0.25% Trypsin | Gibco | 25200072 |
PBS Buffer Solution | Gibco | 10010023 |
References
[1] Ng CE, Inch WR. Comparison of the densities of clonogenic cells from EMT6 fibrosarcoma monolayer cultures, multicell spheroids, and solid tumors in ficoll density gradients. J Natl Cancer Inst. 1978 May;60(5):1017-22.
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