标签归档：organoid

bioGenous类器官基质胶 Organoid Culture ECM (Reduced Growth Factor)(M315066)

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类器官基质胶 Organoid Culture ECM (Reduced Growth  Factor)

货号：M315066

规格：10mL

品牌：bioGenous

产品介绍

Product Description:

Basement membranes are thin continuous layers of specialized extracellular matrices that form the interface on which epithelial, endothelial, or neuronal cells grow. Basement membranes also support muscle and Schwann cells of the peripheral nerves as they are found surrounding these cells. The bioGenousTM Organoid Culture ECM is a soluble form of basement membrane extracted and purified from the Engelbreth-Holm-Swarm (EHS) tumor. The major constituents of the bioGenousTM Organoid Culture ECM include laminin, collagen IV, entactin/nidogen, and heparin sulfate proteoglycan.

bioGenousTM Organoid Culture ECM is a reduced growth factor hydrogel that allows the complete control of the organoid culture microenvironment to allow for more consistent cell growth and reproducible experiments. It supports the growth and maintenance of human embryonic stem cells (hESCs) and tissue-derived stem cells, and for the in vivo study of both healthy and tumor organoids derived from various organs. bioGenousTM Organoid Culture ECM is compatible with all culture media and gels at temperatures above 10°C to form a reconstituted basement membrane.

技术参数

Product Information:

Component

Cat#

Volume

Concentration

Storage& Stability  

bioGenousTM Organoid Culture ECM (Reduced Growth Factor)

M315066

10ml

 

8.5-9.0 mg/mL

-20℃，24 months

 

CAUTION: bioGenousTM Organoid Culture ECM will start to gel if kept above 10°C for an extended period. We recommend that you make aliquots and store at –80°C to –20°C to avoid repeated freeze-thaw cycles.

Directions for Use

1.    Start by thawing the bioGenousTM Organoid Culture ECM by submerging the vial in ice in a 4°C refrigerator, overnight.

2.    After the bioGenousTM Organoid Culture ECM has been completely thawed, gently swirl the vial to carefully mix the content of the vail on ice at all times.

3.    If working with small volumes make aliquots of the bioGenousTM Organoid Culture ECM by gently pipetting it into pre-cold tubes on ice and immediately store the remaining at -20°C.

Note: All cultureware including pipette and centrifuge tubes used should be pre-chilled/ice-cold to avoid the bioGenousTM Organoid Culture ECM from clogging. However, if clogging occurs, the matrix may be re-liquified by placing it at 4°C in ice for 24-48 hours.

4.    bioGenousTM Organoid Culture ECM can be used at a recommended dilution ratio of >70% (ECM: culture medium = 7:3).

Note: Excessive dilution of bioGenousTM Organoid Culture ECM below 50% dilution may result in an extremely thin and fragile non-gelled protein layer that cannot support continuous organoid growth.

5.    Dispense the required volumes of the matrix-cell mixture in the well.

6.    Incubate the plates at 37°C for 15-20 mins and then add the appropriate volume of a pre-warmed growth medium for the specific organoid type.

Directions on Coating with bioGenousTM Organoid Culture ECM

bioGenousTM Organoid Culture ECM (Reduced Growth Factor) could be used in the preparation of thin Gel, thick gel, or thin coating. Thin Gel preparations allow cells to be cultured on top of the gel while the Thick Gel enables the growth of cells within a three-dimensional matrix. Thin Coating could also be done to support cell growth on top of a complex protein layer.

 

Thin Gel
1. Start by thawing the required amount of bioGenousTM Organoid Culture ECM.

2. Gently mix the bioGenousTM Organoid Culture ECM. Be careful not to introduce bubbles into the gel.

3. Dispense 50 µl/cm2 of growth surface into the pre-chilled culture plate.

4. Incubate the plates at 37°C for 30 minutes before use.

5. Aspirate any unbound gel on the surface of the plate and rinse once with the basal organoid medium.

 

Thick Gel

1. Start by thawing the required amount of bioGenousTM Organoid Culture ECM.

2. Gently mix the bioGenousTM Organoid Culture ECM. Be careful not to introduce bubbles into the gel.

3. Add the recommended amount of cells to the bioGenousTM Organoid Culture ECM on ice and dispense 150-200 µl/cm2 of growth surface.

4. Incubate the plates at 37°C for 30 minutes before use.

5. Add culture medium and incubate at 37°C.  

 

Thin Coating Method

1. Start by thawing the required amount of bioGenousTM Organoid Culture ECM.

2. Gently mix the bioGenousTM Organoid Culture ECM. Be careful not to introduce bubbles into the gel.

3. Using the basal organoid medium, dilute the bioGenousTM Organoid Culture ECM. It is necessary to optimize the dilution media to determine the optimal dilution for your investigation.

4. Dispense enough diluted bioGenousTM Organoid Culture ECM to cover the entire growth surface.   

5. Incubate the plates at room temperature for 60 minutes.

6. If any unbound materials remain, aspirate and rinse gently using the basal organoid medium.

 

NB: If gel-coated plates are not used immediately after coating, add PBS to prevent evaporation and store at 4°C for up to 5-10 days.

bioGenous类器官传代消化液 Organoid Dissociation Solution（E238001）

发表于由

Organoid Dissociation Solution 类器官传代消化液

货号：E238001

规格：100ml

500ml

品牌：bioGenous

产品介绍

bioGenousTM Organoid Dissociation Solution（类器官传代消化液）可应用于多种哺乳动物（如人、鼠、猪、蝙蝠、牛等）来源类器官的常规传代，可使类器官从基质胶中分离，并可使其温和地消化为小细胞簇或单细胞，同时保持其传代后的生长活力。

技术参数

产品信息

产品组成

货号

规格

储存条件及周期

bioGenousTM Organoid Dissociation Solution

E238001

100mL/500mL

2-8°C，12 个月

类器官消化过程中可能应用到的其他试剂

试剂

推荐货号

 Organoid Basal Medium

B213152 & B213151

Fetal Bovine Serum, FBS

–

类器官的消化传代

1.         向回收后的类器官中加入 5-10倍类器官基质胶混合物体积的Organoid Dissociation Solution（类器官传代消化液），吹打混匀后在37°C条件下孵育1-8分钟使类器官解离（需提前取所需体积的消化液在37℃条件下预热，通常单层结构类器官的消化时间为0.5-3分钟，多层或者体积较大的类器官消化时间为3-8分钟）。

注意：在此操作过程中须仔细监测消化过程，避免过度消化。在消化过程中，可使用移液器吹打混匀帮助消化。也可实时取少量消化悬液于显微镜下观察消化情况，当观察到较多的单细胞或直径在50μm以下的细胞簇后，即可认为消化完成。

2.         在确认消化完成的悬液中，加入至少五倍体积的类器官基础培养基进行稀释，从而终止消化作用。

注意：时间较长的消化过程结束后可适当加入胎牛血清（Fetal Bovine Serum, FBS）至终浓度2%-5%以保证消化后细胞的活力。

3.         将上步骤所获类器官悬液进行离心（水平离心转子，150-300g，3分钟），弃上清，再次加入基础培养基重悬类器官沉淀。

4.         将上步骤所获类器官悬液进行离心（水平离心转子，150-300g，3分钟），弃上清后所获类器官可用于后续类器官培养、冻存等实验操作。

bioGenous类器官冻存液 Organoid Cryopreservation Medium（Serum Free）（E238023）

发表于由

Organoid Cryopreservation Medium（Serum Free）  类器官冻存液

货号：E238023

规格：100ml

品牌：bioGenous

产品介绍

bioGenousTM Organoid Cryopreservation Medium （类器官冻存液）可应用于多种哺乳动物（如人、鼠、猪、蝙蝠、牛等）来源的类器官和细胞系的低温长期保存，该冻存液不含血清，不含任何动物来源成分，含有 10% DMSO。

技术参数

产品信息

产品组成

货号

规格

储存条件及周期

bioGenousTM Organoid Cryopreservation Medium（Serum Free）

E238023

100mL

4℃，3年

类器官（或细胞）的冻存和复苏

需选用生长状态良好的类器官（或细胞）进行冻存实验。 

类器官（或细胞）的冻存 

1.       向离心后的类器官（或细胞）中加入预冷的（2-8℃）Organoid Cryopreservation Medium（类器官冻存液），吹打混匀后迅速转移至细胞冻存管中（冻存管内细胞悬液体积应大于0.5mL）。

注意：每1mL冻存液推荐冻存1×103-1×107个细胞或对应细胞量的类器官。

2.       将冻存管放入细胞冻存程序降温盒内（降温盒须提前平衡至室温或4℃），随后立即将降温盒放入-80 ℃超低温冰箱中；也可将冻存管进行人工梯度降温处理，如4℃ 静置10分钟，-20℃保存1小时，-80℃保存过夜。

3.       次日或12小时后将冻存管于低温条件下（不高于-70℃，推荐使用干冰或原降温盒）转移至液氮中长期保存。 

类器官（或细胞）的复苏

 4.       提前在37℃条件下预热类器官（或细胞）所需的基础培养基。

5.       在37℃的水浴中快速解冻细胞冻存管，当冻存管内冻存物仅剩些许冰渣残留时立即停止水浴并及时转移至洁净操作台。

6.       将冻存悬液转移至离心管中，缓缓加入5-10倍体积的预热的基础培养基，轻轻混匀。

7.       将上步骤所获类器官（或细胞）悬液进行离心（水平离心转子，150-300g，3分钟），弃上清，再次加入基础培养基重悬类器官（或细胞）沉淀。

8. 将上步骤所获类器官（或细胞）悬液进行离心（水平离心转子，150-300g，3分钟），弃上清后所获类器官（或细胞）可用于后续类器官（或细胞）的培养。

bioGenous肺腺癌 Lung Adenocarcinoma Organoid Kit（K2138-LA）

发表于由

肺腺癌 Lung Adenocarcinoma Organoid Kit

货号：K2138-LA

规格：100ml

500ml

品牌：bioGenous

产品介绍

Product Description:

bioGenousTM Lung Adenocarcinoma Organoid Kit is a chemically defined cell culture medium for the establishment and maintenance of human lung adenocarcinoma organoids. Patient-derived cancer organoids recapitulate the genomic and pathological features of original tumors and therefore hold great promise for medical research and precision medicine.

技术参数

Product Information:

Component

Component Cat#

Volume

Storage & Stability

bioGenousTM Lung Adenocarcinoma Organoid Basal Medium

K2138-LA-A100/A500

100mL/500mL

4℃，12 months

bioGenousTM Lung Adenocarcinoma Organoid Supplement B (50x)

K2138-LA-B100/B500

2mL/10mL

-20℃, avoid repeated freeze-thaw cycles, 12 months

bioGenousTM Lung Adenocarcinoma Organoid Supplement C (250x)

K2138-LA-C100/C500

0.4mL/2mL

-20℃, avoid repeated freeze-thaw cycles, 12 months

Preparation of Lung Adenocarcinoma Organoid Complete Medium

Use a sterile technique to prepare the lung adenocarcinoma organoid complete medium. The following example is for preparing 10mL complete medium. If preparing other volumes, adjust accordingly.

1.      Thaw Lung Adenocarcinoma Organoid Supplement B(50x) and Lung Adenocarcinoma Organoid Supplement C(250x) on ice. Mix thoroughly.

NOTE: Once thawed, use immediately or aliquot and store at -20°C for not more than 10 months. After thawing the aliquots, use immediately. Do not re-freeze.

2.      Add 200μL Lung Adenocarcinoma Organoid Supplement B(50x) and 40μL Lung Adenocarcinoma Organoid Supplement C(250x) to 9.76mL Lung Adenocarcinoma Organoid Basal Medium. Mix thoroughly.

NOTE: If not used immediately, store the complete medium at 2-8°C for not more than 2 weeks. The Lung Adenocarcinoma Organoid Supplement B contains fungicide and antibiotics (50x).

Protocol for Establishment of Patient-Derived Lung Adenocarcinoma Organoids

CAUTION Studies involving primary human tissue material must follow all relevant institutional and government regulations. Informed consent must be obtained from all subjects before the collection of the primary human tissue material.

Establishment of Organoids from Primary Tissue

1.      Collect primary human lung adenocarcinoma tissue pieces in ice-cold Primary Tissue Storage Solution (K601005) with conical tubes. Keep tissue samples at 4°C until the start of the isolation.

2.      Assess whether the obtained tissue pieces consist purely of epithelium. If fat or muscle tissues are present, remove these non-epithelial components as much as possible using surgical scissors or scalpels and forceps under a dissection microscope. If no fat or muscle tissues are present, continue to the next step immediately.

3.      Rinse the tissue with Cancer Organoid Basal Medium (B213152) or DPBS twice.

4.      Mince the tissue into small fragments of 1-3 mm3 in a cell culture dish using surgical scissors or scalpels.

5.      Digest the tissue fragments with 10mL of Tumor Tissue Digestion Solution (K601003) in a 15mL conical tube at 37°C, with variable incubation times ranging from 30 min to 1.5 h. Carefully monitor the digestion process, mixing the content of the tube every 5-10 min by shaking vigorously or pipetting the mixture up and down. The digestion process could be finished when most of tissue fragments are able to pass through the 1mL pipette tips.

6.      Add FBS to the tissue digestion mixture to a final concentration of 2%, and filter using a 100 μm cell strainer.

7.      Collect and centrifuge the filtered cells at 250g for 3 min at 4 °C. In case of a visible red pellet, aspirate the supernatant, and resuspend the pellet using 2mL of Red Blood Cell Lysis Solution (E238010) to lyse the erythrocytes at room temperature for 1 min and centrifuge at 250g for 3 min at 4°C.

8.      Aspirate the supernatant and resuspend the pellet in Cancer Organoid Basal Medium, centrifuge at 250g for 3 min at 4°C, repeat this step once more time.

9.      Aspirate the supernatant and resuspend the pellet in Matrigel. The Matrigel should be kept on ice to prevent it from solidifying. Perform the process as quickly as possible. The amount of Matrigel used depends on the size of the pellet. Approximately 10,000 cells should be plated in 25 μL of Matrigel.

CRITICAL: Do not overly dilute the Matrigel (Matrigel should be >70% (Matrigel vol/Total vol)), as this may inhibit the proper formation of the solid droplets.

10.   Plate the Matrigel containing organoids at the bottom of 24-well cell culture plates in droplets of ~30 μL each around the center of the well..

CRITICAL: Once the organoids are resuspended in Matrigel, proceed with plating as quickly as possible, as the Matrigel may solidify in the tube or pipette tip. Do not let the Matrigel touch the wall of wells.

11.   Place the culture plate into a humidified incubator at 37 °C and 5% (vol/vol) CO2 for 15-25 min to let the Matrigel solidify.

12.   Prepare the required amount of lung adenocarcinoma organoid complete medium.

13.   Once the Matrigel droplets have solidified (15-25 min), open the plate and carefully add 500 μL of organoid complete medium to each well.

CRITICAL: Do not add the medium directly on top of the Matrigel droplets, as this might disrupt the droplets.

14.   Place the culture plate in a humidified incubator at 37 °C and 5% (vol/vol) CO2.

15.   Change the medium every 3-4 d by carefully aspirating the medium from the wells and replacing it with a fresh, pre-warmed organoid complete medium.

16.   Closely monitor the organoid formation. Ideally, patient-derived lung adenocarcinoma organoids should be passaged for the first time between 7 and 10 d after the initial plating.

Splitting and Passaging of Organoids

17.   Pipette up and down to disrupt the Matrigel and transfer the organoid suspension to a 1.5 mL conical tube.

18.   Pipette the organoid suspension up and down to mix thoroughly by pipetting against the bottom of the tube to create pressure, which will aid the removal of Matrigel.

19.   Centrifuge organoids at 250g for 3 min at room temperature.

20.   Aspirate the supernatant and split organoids using either Organoid Dissociation Solution (E238001) or by mechanical disruption. For Organoid Dissociation Solution-based cell dissociation, resuspend the pellet in 0.2 mL of Organoid Dissociation Solution, pipette up and down and incubate at 37 °C until organoids fall apart. Pipette up and down with a filter tip for ≥8 times every 2 min to aid in the disruption of the organoids. Closely monitor the digestion to keep the incubation time in the Organoid Dissociation Solution to a minimum. In case of mechanical disruption, resuspend the pellet in 1.5 mL of Cancer Organoid Basal Medium. Carefully pipette the organoid suspension up and down 30 times by pipetting against the bottom of the tube to create pressure, which will aid organoid disruption.

CRITICAL: Do not dissociate in Organoid Dissociation Solution for >7 min, as this may result in poor organoid outgrowth or even loss of the culture. As a rule of thumb, digestion is complete if a mixture of small lumps of cells (consisting of 10–50 cells) can be observed.

21.   After shearing is complete, wash once by topping up with 1 mL of Cancer Organoid Basal Medium, and centrifuge at 250g for 3 min at room temperature.

22.   Aspirate the supernatant and resuspend the organoid pellet in 70% (vol/vol) Matrigel, and plate organoids in droplets at the bottom of a culture plate as described in Steps 10. After plating is complete, transfer the plate into a humidified incubator at 37 °C and 5% (vol/vol) CO2 for 15–25 min.

23.   Pre-warm lung adenocarcinoma organoid complete medium at 37 °C.

24.   After the Matrigel droplets have solidified (15–25 min), carefully pipette pre-warmed medium into the wells.

25.   Place culture plates in a humidified incubator at 37 °C and 5% (vol/vol) CO2 until the organoids are needed for further experiments.

bioGenous肝内胆管癌 Cholangiocarcinoma Organoid Kit（K2104-LB）

发表于由

肝内胆管癌 Cholangiocarcinoma Organoid Kit

货号：K2104-LB

规格：100ml

500ml

品牌：bioGenous

产品介绍

Product Description:

bioGenousTM Cholangiocarcinoma Organoid Kit is a chemically defined cell culture medium for the establishment and maintenance of human cholangiocarcinoma organoids. Patient-derived cancer organoids recapitulate the genomic and pathological features of original tumors and therefore hold great promise for medical research and precision medicine.

技术参数

Product Information:

Component

Component Cat#

Volume

Storage & Stability

bioGenousTM Cholangiocarcinoma Organoid Basal Medium

K2104-LB-A100/A500

100mL/500mL

4℃，12 months

bioGenousTM Cholangiocarcinoma Organoid Supplement B (50x)

K2104-LB-B100/B500

2mL/10mL

-20℃, avoid repeated freeze-thaw cycles, 12 months

bioGenousTM Cholangiocarcinoma Organoid Supplement C (250x)

K2104-LB-C100/C500

0.4mL/2mL

-20℃, avoid repeated freeze-thaw cycles, 12 months

Preparation of Cholangiocarcinoma Organoid Complete Medium

Use a sterile technique to prepare the cholangiocarcinoma organoid complete medium. The following example is for preparing 10mL complete medium. If preparing other volumes, adjust accordingly.

1.    Thaw Cholangiocarcinoma Organoid Supplement B(50x) and Cholangiocarcinoma Organoid Supplement C(250x) on ice. Mix thoroughly.

NOTE: Once thawed, use immediately or aliquot and store at -20°C for not more than 10 months. After thawing the aliquots, use immediately. Do not re-freeze.

2.      Add 200μL Cholangiocarcinoma Organoid Supplement B(50x) and 40μL Cholangiocarcinoma Organoid Supplement C(250x) to 9.76mL Cholangiocarcinoma Organoid Basal Medium. Mix thoroughly.

NOTE: If not used immediately, store the complete medium at 2-8°C for not more than 2 weeks. The Cholangiocarcinoma Organoid Supplement B contains fungicide and antibiotics (50x).

Protocol for Establishment of Patient-Derived Cholangiocarcinoma Organoids

CAUTION Studies involving primary human tissue material must follow all relevant institutional and government regulations. Informed consent must be obtained from all subjects before the collection of the primary human tissue material.

Establishment of Organoids from Primary Tissue

1.      Collect primary human cholangiocarcinoma tissue pieces in ice-cold Primary Tissue Storage Solution (K601005) with conical tubes. Keep tissue samples at 4°C until the start of the isolation.

2.      Assess whether the obtained tissue pieces consist purely of epithelium. If fat or muscle tissues are present, remove these non-epithelial components as much as possible using surgical scissors or scalpels and forceps under a dissection microscope. If no fat or muscle tissues are present, continue to the next step immediately.

3.      Rinse the tissue with Cancer Organoid Basal Medium (B213152) or DPBS twice.

4.      Mince the tissue into small fragments of 1-3 mm3 in a cell culture dish using surgical scissors or scalpels.

5.      Digest the tissue fragments with 10mL of Tumor Tissue Digestion Solution (K601003) in a 15mL conical tube at 37°C, with variable incubation times ranging from 30 min to 1.5 h. Carefully monitor the digestion process, mixing the content of the tube every 5-10 min by shaking vigorously or pipetting the mixture up and down. The digestion process could be finished when most of tissue fragments are able to pass through the 1mL pipette tips.

6.      Add FBS to the tissue digestion mixture to a final concentration of 2%, and filter using a 100 μm cell strainer.

7.      Collect and centrifuge the filtered cells at 250g for 3 min at 4 °C. In case of a visible red pellet, aspirate the supernatant, and resuspend the pellet using 2mL of Red Blood Cell Lysis Solution (E238010) to lyse the erythrocytes at room temperature for 1 min and centrifuge at 250g for 3 min at 4°C.

8.      Aspirate the supernatant and resuspend the pellet in Cancer Organoid Basal Medium, centrifuge at 250g for 3 min at 4°C, repeat this step once more time.

9.      Aspirate the supernatant and resuspend the pellet in Matrigel. The Matrigel should be kept on ice to prevent it from solidifying. Perform the process as quickly as possible. The amount of Matrigel used depends on the size of the pellet. Approximately 10,000 cells should be plated in 25 μL of Matrigel.

CRITICAL: Do not overly dilute the Matrigel (Matrigel should be >70% (Matrigel vol/Total vol)), as this may inhibit the proper formation of the solid droplets.

10.   Plate the Matrigel containing organoids at the bottom of 24-well cell culture plates in droplets of ~30 μL each around the center of the well..

CRITICAL: Once the organoids are resuspended in Matrigel, proceed with plating as quickly as possible, as the Matrigel may solidify in the tube or pipette tip. Do not let the Matrigel touch the wall of wells.

11.   Place the culture plate into a humidified incubator at 37 °C and 5% (vol/vol) CO2 for 15-25 min to let the Matrigel solidify.

12.   Prepare the required amount of cholangiocarcinoma organoid complete medium.

13.   Once the Matrigel droplets have solidified (15-25 min), open the plate and carefully add 500 μL of organoid complete medium to each well.

CRITICAL: Do not add the medium directly on top of the Matrigel droplets, as this might disrupt the droplets.

14.   Place the culture plate in a humidified incubator at 37 °C and 5% (vol/vol) CO2.

15.   Change the medium every 3-4 d by carefully aspirating the medium from the wells and replacing it with a fresh, pre-warmed organoid complete medium.

16.   Closely monitor the organoid formation. Ideally, patient-derived cholangiocarcinoma organoids should be passaged for the first time between 7 and 10 d after the initial plating.

Splitting and Passaging of Organoids

17.   Pipette up and down to disrupt the Matrigel and transfer the organoid suspension to a 1.5 mL conical tube.

18.   Pipette the organoid suspension up and down to mix thoroughly by pipetting against the bottom of the tube to create pressure, which will aid the removal of Matrigel.

19.   Centrifuge organoids at 250g for 3 min at room temperature.

20.   Aspirate the supernatant and split organoids using either Organoid Dissociation Solution (E238001) or by mechanical disruption. For Organoid Dissociation Solution-based cell dissociation, resuspend the pellet in 0.2 mL of Organoid Dissociation Solution, pipette up and down and incubate at 37 °C until organoids fall apart. Pipette up and down with a filter tip for ≥8 times every 2 min to aid in the disruption of the organoids. Closely monitor the digestion to keep the incubation time in the Organoid Dissociation Solution to a minimum. In case of mechanical disruption, resuspend the pellet in 1.5 mL of Cancer Organoid Basal Medium. Carefully pipette the organoid suspension up and down 30 times by pipetting against the bottom of the tube to create pressure, which will aid organoid disruption.

CRITICAL: Do not dissociate in Organoid Dissociation Solution for >5 min, as this may result in poor organoid outgrowth or even loss of the culture. As a rule of thumb, digestion is complete if a mixture of small lumps of cells (consisting of 10–50 cells) can be observed.

21.   After shearing is complete, wash once by topping up with 1 mL of Cancer Organoid Basal Medium, and centrifuge at 250g for 3 min at room temperature.

22.   Aspirate the supernatant and resuspend the organoid pellet in 70% (vol/vol) Matrigel, and plate organoids in droplets at the bottom of a culture plate as described in Steps 10. After plating is complete, transfer the plate into a humidified incubator at 37 °C and 5% (vol/vol) CO2 for 15–25 min.

23.   Pre-warm cholangiocarcinoma organoid complete medium at 37 °C.

24.   After the Matrigel droplets have solidified (15–25 min), carefully pipette pre-warmed medium into the wells.

Place culture plates in a humidified incubator at 37 °C and 5% (vol/vol) CO2 until the organoids are needed for further experiments.

bioGenous肝实质细胞癌 Hepatocellular Carcinoma Organoid Kit（K2105-HCC）

发表于由

肝实质细胞癌 Hepatocellular Carcinoma Organoid Kit

货号：K2105-HCC

规格：100ml

500ml

品牌：bioGenous

产品介绍

Product Description:

bioGenousTM Hepatocellular Carcinoma Organoid Kit is a chemically defined cell culture medium for the establishment and maintenance of human hepatocellular carcinoma organoids. Patient-derived cancer organoids recapitulate the genomic and pathological features of original tumors and therefore hold great promise for medical research and precision medicine.

技术参数

Product Information:

Component

Component Cat#

Volume

Storage & Stability

bioGenousTM Hepatocellular Carcinoma Organoid Basal Medium

K2105-HCC-A100/A500

100mL/500mL

4℃，12 months

bioGenousTM Hepatocellular Carcinoma Organoid Supplement B (50x)

K2105-HCC-B100/B500

2mL/10mL

-20℃, avoid repeated freeze-thaw cycles, 12 months

bioGenousTM Hepatocellular Carcinoma Organoid Supplement C (250x)

K2105-HCC-C100/C500

0.4mL/2mL

-20℃, avoid repeated freeze-thaw cycles, 12 months

Preparation of Hepatocellular Carcinoma Organoid Complete Medium

Use a sterile technique to prepare the hepatocellular carcinoma organoid complete medium. The following example is for preparing 10mL complete medium. If preparing other volumes, adjust accordingly.

1.      Thaw Hepatocellular Carcinoma Organoid Supplement B(50x) and Hepatocellular Carcinoma Organoid Supplement C(250x) on ice. Mix thoroughly.

NOTE: Once thawed, use immediately or aliquot and store at -20°C for not more than 10 months. After thawing the aliquots, use immediately. Do not re-freeze.

2.      Add 200μL Hepatocellular Carcinoma Organoid Supplement B(50x) and 40μL Hepatocellular Carcinoma Organoid Supplement C(250x) to 9.76mL Hepatocellular Carcinoma Organoid Basal Medium. Mix thoroughly.

NOTE: If not used immediately, store the complete medium at 2-8°C for not more than 2 weeks. The Hepatocellular Carcinoma Organoid Supplement B contains fungicide and antibiotics (50x).

Protocol for Establishment of Patient-Derived Hepatocellular Carcinoma Organoids

CAUTION Studies involving primary human tissue material must follow all relevant institutional and government regulations. Informed consent must be obtained from all subjects before the collection of the primary human tissue material.

Establishment of Organoids from Primary Tissue

1.      Collect primary human hepatocellular carcinoma tissue pieces in ice-cold Primary Tissue Storage Solution (K601005) with conical tubes. Keep tissue samples at 4°C until the start of the isolation.

2.      Assess whether the obtained tissue pieces consist purely of epithelium. If fat or muscle tissues are present, remove these non-epithelial components as much as possible using surgical scissors or scalpels and forceps under a dissection microscope. If no fat or muscle tissues are present, continue to the next step immediately.

3.      Rinse the tissue with Cancer Organoid Basal Medium (B213152) or DPBS twice.

4.      Mince the tissue into small fragments of 1-3 mm3 in a cell culture dish using surgical scissors or scalpels.

5.      Digest the tissue fragments with 10mL of Tumor Tissue Digestion Solution (K601003) in a 15mL conical tube at 37°C, with variable incubation times ranging from 15 min to 45 min. Carefully monitor the digestion process, mixing the content of the tube every 5-10 min by shaking vigorously or pipetting the mixture up and down. The digestion process could be finished when most of tissue fragments are able to pass through the 1mL pipette tips.

6.      Add FBS to the tissue digestion mixture to a final concentration of 2%, and filter using a 100 μm cell strainer.

7.      Collect and centrifuge the filtered cells at 250g for 3 min at 4 °C. In case of a visible red pellet, aspirate the supernatant, and resuspend the pellet using 2mL of Red Blood Cell Lysis Solution (E238010) to lyse the erythrocytes at room temperature for 1 min and centrifuge at 250g for 3 min at 4°C.

8.      Aspirate the supernatant and resuspend the pellet in Cancer Organoid Basal Medium, centrifuge at 250g for 3 min at 4°C, repeat this step once more time.

9.      Aspirate the supernatant and resuspend the pellet in Matrigel. The Matrigel should be kept on ice to prevent it from solidifying. Perform the process as quickly as possible. The amount of Matrigel used depends on the size of the pellet. Approximately 10,000 cells should be plated in 25 μL of Matrigel.

CRITICAL: Do not overly dilute the Matrigel (Matrigel should be >70% (Matrigel vol/Total vol)), as this may inhibit the proper formation of the solid droplets.

10.   Plate the Matrigel containing organoids at the bottom of 24-well cell culture plates in droplets of ~30 μL each around the center of the well..

CRITICAL: Once the organoids are resuspended in Matrigel, proceed with plating as quickly as possible, as the Matrigel may solidify in the tube or pipette tip. Do not let the Matrigel touch the wall of wells.

11.   Place the culture plate into a humidified incubator at 37 °C and 5% (vol/vol) CO2 for 15-25 min to let the Matrigel solidify.

12.   Prepare the required amount of hepatocellular carcinoma organoid complete medium.

13.   Once the Matrigel droplets have solidified (15-25 min), open the plate and carefully add 500 μL of organoid complete medium to each well.

CRITICAL: Do not add the medium directly on top of the Matrigel droplets, as this might disrupt the droplets.

14.   Place the culture plate in a humidified incubator at 37 °C and 5% (vol/vol) CO2.

15.   Change the medium every 3-4 d by carefully aspirating the medium from the wells and replacing it with a fresh, pre-warmed organoid complete medium.

16.   Closely monitor the organoid formation. Ideally, patient-derived hepatocellular carcinoma organoids should be passaged for the first time between 7 and 10 d after the initial plating.

Splitting and Passaging of Organoids

17.   Pipette up and down to disrupt the Matrigel and transfer the organoid suspension to a 1.5 mL conical tube.

18.   Pipette the organoid suspension up and down to mix thoroughly by pipetting against the bottom of the tube to create pressure, which will aid the removal of Matrigel.

19.   Centrifuge organoids at 250g for 3 min at room temperature.

20.   Aspirate the supernatant and split organoids using either Organoid Dissociation Solution (E238001) or by mechanical disruption. For Organoid Dissociation Solution-based cell dissociation, resuspend the pellet in 0.2 mL of Organoid Dissociation Solution, pipette up and down and incubate at 37 °C until organoids fall apart. Pipette up and down with a filter tip for ≥5 times every 1 min to aid in the disruption of the organoids. Closely monitor the digestion to keep the incubation time in the Organoid Dissociation Solution to a minimum. In case of mechanical disruption, resuspend the pellet in 1.5 mL of Cancer Organoid Basal Medium. Carefully pipette the organoid suspension up and down 30 times by pipetting against the bottom of the tube to create pressure, which will aid organoid disruption.

CRITICAL: Do not dissociate in Organoid Dissociation Solution for >3 min, as this may result in poor organoid outgrowth or even loss of the culture. As a rule of thumb, digestion is complete if a mixture of small lumps of cells (consisting of 10–50 cells) can be observed.

21.   After shearing is complete, wash once by topping up with 1 mL of Cancer Organoid Basal Medium, and centrifuge at 250g for 3 min at room temperature.

22.   Aspirate the supernatant and resuspend the organoid pellet in 70% (vol/vol) Matrigel, and plate organoids in droplets at the bottom of a culture plate as described in Steps 10. After plating is complete, transfer the plate into a humidified incubator at 37 °C and 5% (vol/vol) CO2 for 15–25 min.

23.   Pre-warm hepatocellular carcinoma organoid complete medium at 37 °C.

24.   After the Matrigel droplets have solidified (15–25 min), carefully pipette pre-warmed medium into the wells.

25.   Place culture plates in a humidified incubator at 37 °C and 5% (vol/vol) CO2 until the organoids are needed for further experiments.

bioGenous宫颈癌 Cervical Cancer Organoid Kit（K2169-CC）

发表于由

宫颈癌 Cervical Cancer Organoid Kit

货号：K2169-CC

规格：100ml

500ml

品牌：bioGenous

产品介绍

Product Description:

bioGenousTM Cervical Cancer Organoid Kit is a chemically defined cell culture medium for the establishment and maintenance of human cervical cancer organoids. Patient-derived cancer organoids recapitulate the genomic and pathological features of original tumors and therefore hold great promise for medical research and precision medicine.

技术参数

Product Information:

Component

Component Cat#

Volume

Storage & Stability

bioGenousTM Cervical Cancer Organoid Basal Medium

K2169-CC-A100/A500

100mL/500mL

4℃，12 months

bioGenousTM Cervical Cancer Organoid Supplement B (50x)

K2169-CC-B100/B500

2mL/10mL

-20℃, avoid repeated freeze-thaw cycles, 12 months

bioGenousTM Cervical Cancer Organoid Supplement C (250x)

K2169-CC-C100/C500

0.4mL/2mL

-20℃, avoid repeated freeze-thaw cycles, 12 months

Preparation of Cervical Cancer Organoid Complete Medium

Use a sterile technique to prepare the cervical cancer organoid complete medium. The following example is for preparing 10mL complete medium. If preparing other volumes, adjust accordingly.

1.      Thaw Cervical Cancer Organoid Supplement B(50x) and Cervical Cancer Organoid Supplement C(250x) on ice. Mix thoroughly.

NOTE: Once thawed, use immediately or aliquot and store at -20°C for not more than 10 months. After thawing the aliquots, use immediately. Do not re-freeze.

2.      Add 200μL Cervical Cancer Organoid Supplement B(50x) and 40μL Cervical Cancer Organoid Supplement C(250x) to 9.76mL Cervical Cancer Organoid Basal Medium. Mix thoroughly.

NOTE: If not used immediately, store the complete medium at 2-8°C for not more than 2 weeks. The Cervical Cancer Organoid Supplement B contains fungicide and antibiotics (50x).

Protocol for Establishment of Patient-Derived Cervical Cancer Organoids

CAUTION Studies involving primary human tissue material must follow all relevant institutional and government regulations. Informed consent must be obtained from all subjects before the collection of the primary human tissue material.

Establishment of Organoids from Primary Tissue

1.      Collect primary human cervical cancer tissue pieces in ice-cold Primary Tissue Storage Solution (K601005) with conical tubes. Keep tissue samples at 4°C until the start of the isolation.

2.      Assess whether the obtained tissue pieces consist purely of epithelium. If fat or muscle tissues are present, remove these non-epithelial components as much as possible using surgical scissors or scalpels and forceps under a dissection microscope. If no fat or muscle tissues are present, continue to the next step immediately.

3.      Rinse the tissue with Cancer Organoid Basal Medium (B213152) or DPBS twice.

4.      Mince the tissue into small fragments of 1-3 mm3 in a cell culture dish using surgical scissors or scalpels.

5.      Digest the tissue fragments with 10mL of Tumor Tissue Digestion Solution (K601003) in a 15mL conical tube at 37°C, with variable incubation times ranging from 30 min to 1 h. Carefully monitor the digestion process, mixing the content of the tube every 5-10 min by shaking vigorously or pipetting the mixture up and down. The digestion process could be finished when most of tissue fragments are able to pass through the 1mL pipette tips.

6.      Add FBS to the tissue digestion mixture to a final concentration of 2%, and filter using a 100 μm cell strainer.

7.      Collect and centrifuge the filtered cells at 250g for 3 min at 4 °C. In case of a visible red pellet, aspirate the supernatant, and resuspend the pellet using 2mL of Red Blood Cell Lysis Solution (E238010) to lyse the erythrocytes at room temperature for 1 min and centrifuge at 250g for 3 min at 4°C.

8.      Aspirate the supernatant and resuspend the pellet in Cancer Organoid Basal Medium, centrifuge at 250g for 3 min at 4°C, repeat this step once more time.

9.      Aspirate the supernatant and resuspend the pellet in Matrigel. The Matrigel should be kept on ice to prevent it from solidifying. Perform the process as quickly as possible. The amount of Matrigel used depends on the size of the pellet. Approximately 10,000 cells should be plated in 25 μL of Matrigel.

CRITICAL: Do not overly dilute the Matrigel (Matrigel should be >70% (Matrigel vol/Total vol)), as this may inhibit the proper formation of the solid droplets.

10.   Plate the Matrigel containing organoids at the bottom of 24-well cell culture plates in droplets of ~30 μL each around the center of the well..

CRITICAL: Once the organoids are resuspended in Matrigel, proceed with plating as quickly as possible, as the Matrigel may solidify in the tube or pipette tip. Do not let the Matrigel touch the wall of wells.

11.   Place the culture plate into a humidified incubator at 37 °C and 5% (vol/vol) CO2 for 15-25 min to let the Matrigel solidify.

12.   Prepare the required amount of cervical cancer organoid complete medium.

13.   Once the Matrigel droplets have solidified (15-25 min), open the plate and carefully add 500 μL of organoid complete medium to each well.

CRITICAL: Do not add the medium directly on top of the Matrigel droplets, as this might disrupt the droplets.

14.   Place the culture plate in a humidified incubator at 37 °C and 5% (vol/vol) CO2.

15.   Change the medium every 3-4 d by carefully aspirating the medium from the wells and replacing it with a fresh, pre-warmed organoid complete medium.

16.   Closely monitor the organoid formation. Ideally, patient-derived cervical cancer organoids should be passaged for the first time between 7 and 10 d after the initial plating.

Splitting and Passaging of Organoids

17.   Pipette up and down to disrupt the Matrigel and transfer the organoid suspension to a 1.5 mL conical tube.

18.   Pipette the organoid suspension up and down to mix thoroughly by pipetting against the bottom of the tube to create pressure, which will aid the removal of Matrigel.

19.   Centrifuge organoids at 250g for 3 min at room temperature.

20.   Aspirate the supernatant and split organoids using either Organoid Dissociation Solution (E238001) or by mechanical disruption. For Organoid Dissociation Solution-based cell dissociation, resuspend the pellet in 0.2 mL of Organoid Dissociation Solution, pipette up and down and incubate at 37 °C until organoids fall apart. Pipette up and down with a filter tip for ≥8 times every 2 min to aid in the disruption of the organoids. Closely monitor the digestion to keep the incubation time in the Organoid Dissociation Solution to a minimum. In case of mechanical disruption, resuspend the pellet in 1.5 mL of Cancer Organoid Basal Medium. Carefully pipette the organoid suspension up and down 30 times by pipetting against the bottom of the tube to create pressure, which will aid organoid disruption.

CRITICAL: Do not dissociate in Organoid Dissociation Solution for >7 min, as this may result in poor organoid outgrowth or even loss of the culture. As a rule of thumb, digestion is complete if a mixture of small lumps of cells (consisting of 10–50 cells) can be observed.

21.   After shearing is complete, wash once by topping up with 1 mL of Cancer Organoid Basal Medium, and centrifuge at 250g for 3 min at room temperature.

22.   Aspirate the supernatant and resuspend the organoid pellet in 70% (vol/vol) Matrigel, and plate organoids in droplets at the bottom of a culture plate as described in Steps 10. After plating is complete, transfer the plate into a humidified incubator at 37 °C and 5% (vol/vol) CO2 for 15–25 min.

23.   Pre-warm cervical cancer organoid complete medium at 37 °C.

24.   After the Matrigel droplets have solidified (15–25 min), carefully pipette pre-warmed medium into the wells.

25.   Place culture plates in a humidified incubator at 37 °C and 5% (vol/vol) CO2 until the organoids are needed for further experiments.

bioGenous结直肠癌 Colorectal Cancer Organoid Kit（K2103-CR）

发表于由

结直肠癌 Colorectal Cancer Organoid Kit

货号：K2103-CR

规格：100ml

500ml

品牌：bioGenous

产品介绍

Product Description:

bioGenousTM Colorectal Cancer Organoid Kit is a chemically defined cell culture medium for the establishment and maintenance of human Colorectal cancer organoids. Patient-derived cancer organoids recapitulate the genomic and pathological features of original tumors and therefore hold great promise for medical research and precision medicine.

技术参数

Product Information:

Component

Component Cat#

Volume

Storage& Stability

bioGenousTM Colorectal Cancer Organoid Basal Medium

K2103-CR-A100/A500

100mL/500mL

4℃，12 months

bioGenousTM Colorectal Cancer Organoid Supplement B (50x)

K2103-CR-B100/B500

2mL/10mL

-20℃,avoid repeated freeze-thaw cycles, 12 months

bioGenousTM Colorectal Cancer Organoid Supplement C (250x)

K2103-CR-C100/C500

0.4mL/2mL

-20℃, avoid repeated freeze-thaw cycles, 12 months

Preparation of Colorectal Cancer Organoid Complete Medium

Use a sterile technique to prepare the colorectal cancer organoid complete medium. The following example is for preparing 10mL complete medium. If preparing other volumes, adjust accordingly.

1.       Thaw Colorectal Cancer Organoid Supplement B(50x) and Colorectal Cancer Organoid Supplement C(250x) on ice. Mix thoroughly.

NOTE: Once thawed, use immediately or aliquot and store at -20°C for not more than 10 months. After thawing the aliquots, use immediately. Do not re-freeze.

2.       Add 200μL Colorectal Cancer Organoid Supplement B(50x) and 40μL Colorectal Cancer Organoid Supplement C(250x) to 9.76mL Colorectal Cancer Organoid Basal Medium. Mix thoroughly.

NOTE: If not used immediately, store the complete medium at 2-8°C for not more than 2 weeks. The Colorectal Cancer Organoid Supplement B contains fungicide and antibiotics (50x).

Protocol for Establishment of Patient-Derived Colorectal Cancer Organoids

CAUTIONStudies involving primary human tissue material must follow all relevant institutional and government regulations. Informed consent must be obtained from all subjects before the collection of the primary human tissue material.

Establishment of Organoids from Primary Tissue

1.       Collect primary human colorectal cancer tissue pieces in ice-cold Primary Tissue Storage Solution (K601005) with conical tubes. Keep tissue samples at 4°C until the start of the isolation.

2.       Assess whether the obtained tissue pieces consist purely of epithelium. If fat or muscle tissues are present, remove these non-epithelial components as much as possible using surgical scissors or scalpels and forceps under a dissection microscope. If no fat or muscle tissues are present, continue to the next step immediately.

3.       Rinse the tissue with Cancer Organoid Basal Medium (B213152) or DPBS twice.

4.       Mince the tissue into small fragments of 1-3 mm3 in a cell culture dish using surgical scissors or scalpels.

5.       Digest the tissue fragments with 10mL of Tumor Tissue Digestion Solution (K601003) in a 15mL conical tube at 37°C, with variable incubation times ranging from 30 min to 1.5 h. Carefully monitor the digestion process, mixing the content of the tube every 5-10 min by shaking vigorously or pipetting the mixture up and down. The digestion process could be finished when most of tissue fragments are able to pass through the 1mL pipette tips.

6.       Add FBS to the tissue digestion mixture to a final concentration of 2%, and filter using a 100 μm cell strainer.

7.       Collect and centrifuge the filtered cells at 250g for 3 min at 4 °C. In case of a visible red pellet, aspirate the supernatant, and resuspend the pellet using 2mL of Red Blood Cell Lysis Solution (E238010) to lyse the erythrocytes at room temperature for 1 min and centrifuge at 250g for 3 min at 4°C.

8.       Aspirate the supernatant and resuspend the pellet in Cancer Organoid Basal Medium, centrifuge at 250g for 3 min at 4°C, repeat this step once more time.

9.       Aspirate the supernatant and resuspend the pellet in Matrigel. The Matrigel should be kept on ice to prevent it from solidifying. Perform the process as quickly as possible. The amount of Matrigel used depends on the size of the pellet. Approximately 10,000 cells should be plated in 25 μL of Matrigel.

CRITICAL: Do not overly dilute the Matrigel (Matrigel should be >70% (Matrigel vol/Total vol)), as this may inhibit the proper formation of the solid droplets.

10.     Plate the Matrigel containing organoids at the bottom of 24-well cell culture plates in droplets of ~30 μL each around the center of the well..

CRITICAL: Once the organoids are resuspended in Matrigel, proceed with plating as quickly as possible, as the Matrigel may solidify in the tube or pipette tip. Do not let the Matrigel touch the wall of wells.

11.     Place the culture plate into a humidified incubator at 37 °C and 5% (vol/vol) CO2 for 15-25 min to let the Matrigel solidify.

12.     Prepare the required amount of colorectal cancer organoid complete medium.

13.     Once the Matrigel droplets have solidified (15-25 min), open the plate and carefully add 500 μL of organoid complete medium to each well.

CRITICAL: Do not add the medium directly on top of the Matrigel droplets, as this might disrupt the droplets.

14.     Place the culture plate in a humidified incubator at 37 °C and 5% (vol/vol) CO2.

15.     Change the medium every 3-4 d by carefully aspirating the medium from the wells and replacing it with a fresh, pre-warmed organoid complete medium.

16.     Closely monitor the organoid formation. Ideally, patient-derived colorectal cancer organoids should be passaged for the first time between 7 and 10 d after the initial plating.

Splitting and Passaging of Organoids

17.     Pipette up and down to disrupt the Matrigel and transfer the organoid suspension to a 1.5 mL conical tube.

18.     Pipette the organoid suspension up and down to mix thoroughly by pipetting against the bottom of the tube to create pressure, which will aid the removal of Matrigel.

19.     Centrifuge organoids at 250g for 3 min at room temperature.

20.     Aspirate the supernatant and split organoids using either Organoid Dissociation Solution (E238001) or by mechanical disruption. For Organoid Dissociation Solution-based cell dissociation, resuspend the pellet in 0.2 mL of Organoid Dissociation Solution, pipette up and down and incubate at 37 °C until organoids fall apart. Pipette up and down with a filter tip for ≥8 times every 2 min to aid in the disruption of the organoids. Closely monitor the digestion to keep the incubation time in the Organoid Dissociation Solution to a minimum. In case of mechanical disruption, resuspend the pellet in 1.5 mL of Cancer Organoid Basal Medium. Carefully pipette the organoid suspension up and down 30 times by pipetting against the bottom of the tube to create pressure, which will aid organoid disruption.

CRITICAL: Do not dissociate in Organoid Dissociation Solution for >6 min, as this may result in poor organoid outgrowth or even loss of the culture. As a rule of thumb, digestion is complete if a mixture of small lumps of cells (consisting of 10–50 cells) can be observed.

21.     After shearing is complete, wash once by topping up with 1 mL ofCancer Organoid Basal Medium, and centrifuge at 250g for 3 min at room temperature.

22.     Aspirate the supernatant and resuspend the organoid pellet in 70% (vol/vol) Matrigel, and plate organoids in droplets at the bottom of a culture plate as described in Steps 10. After plating is complete, transfer the plate into a humidified incubator at 37 °C and 5% (vol/vol) CO2 for 15–25 min.

23.     Pre-warm colorectal cancer organoid complete medium at 37 °C.

24.     After the Matrigel droplets have solidified (15–25 min), carefully pipette pre-warmed medium into the wells.

Place culture plates in a humidified incubator at 37 °C and 5% (vol/vol) CO2 until the organoids are needed for further experiments.

bioGenous卵巢癌 Ovarian Cancer Organoid Kit（K2168-OC）

发表于由

卵巢癌 Ovarian Cancer Organoid Kit

货号：K2168-OC

规格：100ml

500ml

品牌：bioGenous

产品介绍

Product Description:

bioGenousTM Ovarian Cancer Organoid Kit is a chemically defined cell culture medium for the establishment and maintenance of human Ovarian Cancer organoids. Patient-derived cancer organoids recapitulate the genomic and pathological features of original tumors and therefore hold great promise for medical research and precision medicine.

技术参数

Product Information:

Component

Component Cat#

Volume

Storage & Stability

bioGenousTM Ovarian Cancer Organoid Basal Medium

K2168-OC-A100/A500

100mL/500mL

4℃，12 months

bioGenousTM Ovarian Cancer Organoid Supplement B (50x)

K2168-OC-B100/B500

2mL/10mL

-20℃, avoid repeated freeze-thaw cycles, 12 months

bioGenousTM Ovarian Cancer Organoid Supplement C (250x)

K2168-OC-C100/C500

0.4mL/2mL

-20℃, avoid repeated freeze-thaw cycles, 12 months

Preparation of Ovarian Cancer Organoid Complete Medium

Use a sterile technique to prepare the ovarian cancer organoid complete medium. The following example is for preparing 10mL complete medium. If preparing other volumes, adjust accordingly.

1.      Thaw Ovarian Cancer Organoid Supplement B(50x) and Ovarian Cancer Organoid Supplement C(250x) on ice. Mix thoroughly.

NOTE: Once thawed, use immediately or aliquot and store at -20°C for not more than 10 months. After thawing the aliquots, use immediately. Do not re-freeze.

2.      Add 200μL Ovarian Cancer Organoid Supplement B(50x) and 40μL Ovarian Cancer Organoid Supplement C(250x) to 9.76mL Ovarian Cancer Organoid Basal Medium. Mix thoroughly.

NOTE: If not used immediately, store the complete medium at 2-8°C for not more than 2 weeks. The Ovarian Cancer Organoid Supplement B contains fungicide and antibiotics (50x).

Protocol for Establishment of Patient-Derived Ovarian Cancer Organoids

CAUTION Studies involving primary human tissue material must follow all relevant institutional and government regulations. Informed consent must be obtained from all subjects before the collection of the primary human tissue material.

Establishment of Organoids from Primary Tissue

1.      Collect primary human ovarian cancer tissue pieces in ice-cold Primary Tissue Storage Solution (K601005) with conical tubes. Keep tissue samples at 4°C until the start of the isolation.

2.      Assess whether the obtained tissue pieces consist purely of epithelium. If fat or muscle tissues are present, remove these non-epithelial components as much as possible using surgical scissors or scalpels and forceps under a dissection microscope. If no fat or muscle tissues are present, continue to the next step immediately.

3.      Rinse the tissue with Cancer Organoid Basal Medium (B213152) or DPBS twice.

4.      Mince the tissue into small fragments of 1-3 mm3 in a cell culture dish using surgical scissors or scalpels.

5.      Digest the tissue fragments with 10mL of Tumor Tissue Digestion Solution (K601003) in a 15mL conical tube at 37°C, with variable incubation times ranging from 30 min to 1.5 h. Carefully monitor the digestion process, mixing the content of the tube every 5-10 min by shaking vigorously or pipetting the mixture up and down. The digestion process could be finished when most of tissue fragments are able to pass through the 1mL pipette tips.

6.      Add FBS to the tissue digestion mixture to a final concentration of 2%, and filter using a 100 μm cell strainer.

7.      Collect and centrifuge the filtered cells at 250g for 3 min at 4 °C. In case of a visible red pellet, aspirate the supernatant, and resuspend the pellet using 2mL of Red Blood Cell Lysis Solution (E238010) to lyse the erythrocytes at room temperature for 1 min and centrifuge at 250g for 3 min at 4°C.

8.      Aspirate the supernatant and resuspend the pellet in Cancer Organoid Basal Medium, centrifuge at 250g for 3 min at 4°C, repeat this step once more time.

9.      Aspirate the supernatant and resuspend the pellet in Matrigel. The Matrigel should be kept on ice to prevent it from solidifying. Perform the process as quickly as possible. The amount of Matrigel used depends on the size of the pellet. Approximately 10,000 cells should be plated in 25 μL of Matrigel.

CRITICAL: Do not overly dilute the Matrigel (Matrigel should be >70% (Matrigel vol/Total vol)), as this may inhibit the proper formation of the solid droplets.

10.   Plate the Matrigel containing organoids at the bottom of 24-well cell culture plates in droplets of ~30 μL each around the center of the well..

CRITICAL: Once the organoids are resuspended in Matrigel, proceed with plating as quickly as possible, as the Matrigel may solidify in the tube or pipette tip. Do not let the Matrigel touch the wall of wells.

11.   Place the culture plate into a humidified incubator at 37 °C and 5% (vol/vol) CO2 for 15-25 min to let the Matrigel solidify.

12.   Prepare the required amount of ovarian cancer organoid complete medium.

13.   Once the Matrigel droplets have solidified (15-25 min), open the plate and carefully add 500 μL of organoid complete medium to each well.

CRITICAL: Do not add the medium directly on top of the Matrigel droplets, as this might disrupt the droplets.

14.   Place the culture plate in a humidified incubator at 37 °C and 5% (vol/vol) CO2.

15.   Change the medium every 3-4 d by carefully aspirating the medium from the wells and replacing it with a fresh, pre-warmed organoid complete medium.

16.   Closely monitor the organoid formation. Ideally, patient-derived ovarian cancer organoids should be passaged for the first time between 7 and 10 d after the initial plating.

Splitting and Passaging of Organoids

17.   Pipette up and down to disrupt the Matrigel and transfer the organoid suspension to a 1.5 mL conical tube.

18.   Pipette the organoid suspension up and down to mix thoroughly by pipetting against the bottom of the tube to create pressure, which will aid the removal of Matrigel.

19.   Centrifuge organoids at 250g for 3 min at room temperature.

20.   Aspirate the supernatant and split organoids using either Organoid Dissociation Solution (E238001) or by mechanical disruption. For Organoid Dissociation Solution-based cell dissociation, resuspend the pellet in 0.2 mL of Organoid Dissociation Solution, pipette up and down and incubate at 37 °C until organoids fall apart. Pipette up and down with a filter tip for ≥8 times every 2 min to aid in the disruption of the organoids. Closely monitor the digestion to keep the incubation time in the Organoid Dissociation Solution to a minimum. In case of mechanical disruption, resuspend the pellet in 1.5 mL of Cancer Organoid Basal Medium. Carefully pipette the organoid suspension up and down 30 times by pipetting against the bottom of the tube to create pressure, which will aid organoid disruption.

CRITICAL: Do not dissociate in Organoid Dissociation Solution for >7 min, as this may result in poor organoid outgrowth or even loss of the culture. As a rule of thumb, digestion is complete if a mixture of small lumps of cells (consisting of 10–50 cells) can be observed.

21.   After shearing is complete, wash once by topping up with 1 mL of Cancer Organoid Basal Medium, and centrifuge at 250g for 3 min at room temperature.

22.   Aspirate the supernatant and resuspend the organoid pellet in 70% (vol/vol) Matrigel, and plate organoids in droplets at the bottom of a culture plate as described in Steps 10. After plating is complete, transfer the plate into a humidified incubator at 37 °C and 5% (vol/vol) CO2 for 15–25 min.

23.   Pre-warm ovarian cancer organoid complete medium at 37 °C.

24.   After the Matrigel droplets have solidified (15–25 min), carefully pipette pre-warmed medium into the wells.

25.   Place culture plates in a humidified incubator at 37 °C and 5% (vol/vol) CO2 until the organoids are needed for further experiments.

bioGenous乳腺癌 Breast Cancer Organoid Kit（K2147-BC）

发表于由

乳腺癌 Breast Cancer Organoid Kit

货号：K2147-BC

规格：100ml

500ml

品牌：bioGenous

产品介绍

Product Description:

bioGenousTM Breast Cancer Organoid Kit is a chemically defined cell culture medium for the establishment and maintenance of human breast cancer organoids. Patient-derived cancer organoids recapitulate the genomic and pathological features of original tumors and therefore hold great promise for medical research and precision medicine.

技术参数

Product Information:

Component

Component Cat#

Volume

Storage & Stability

bioGenousTM Breast Cancer Organoid Basal Medium

K2147-BC-A100/A500

100mL/500mL

4℃，12 months

bioGenousTM Breast Cancer Organoid Supplement B (50x)

K2147-BC-B100/B500

2mL/10mL

-20℃, avoid repeated freeze-thaw cycles, 12 months

bioGenousTM Breast Cancer Organoid Supplement C (250x)

K2147-BC-C100/C500

0.4mL/2mL

-20℃, avoid repeated freeze-thaw cycles, 12 months

Preparation of Breast Cancer Organoid Complete Medium

Use a sterile technique to prepare the breast cancer organoid complete medium. The following example is for preparing 10mL complete medium. If preparing other volumes, adjust accordingly.

1.      Thaw Breast Cancer Organoid Supplement B(50x) and Breast Cancer Organoid Supplement C(250x) on ice. Mix thoroughly.

NOTE: Once thawed, use immediately or aliquot and store at -20°C for not more than 10 months. After thawing the aliquots, use immediately. Do not re-freeze.

2.      Add 200μL Breast Cancer Organoid Supplement B(50x) and 40μL Breast Cancer Organoid Supplement C(250x) to 9.76mL Breast Cancer Organoid Basal Medium. Mix thoroughly.

NOTE: If not used immediately, store the complete medium at 2-8°C for not more than 2 weeks. The Breast Cancer Organoid Supplement B contains fungicide and antibiotics (50x).

Protocol for Establishment of Patient-Derived Breast Cancer Organoids

CAUTION Studies involving primary human tissue material must follow all relevant institutional and government regulations. Informed consent must be obtained from all subjects before the collection of the primary human tissue material.

Establishment of Organoids from Primary Tissue

1.      Collect primary human breast cancer tissue pieces in ice-cold Primary Tissue Storage Solution (K601005) with conical tubes. Keep tissue samples at 4°C until the start of the isolation.

2.      Assess whether the obtained tissue pieces consist purely of epithelium. If fat or muscle tissues are present, remove these non-epithelial components as much as possible using surgical scissors or scalpels and forceps under a dissection microscope. If no fat or muscle tissues are present, continue to the next step immediately.

3.      Rinse the tissue with Cancer Organoid Basal Medium (B213152) or DPBS twice.

4.      Mince the tissue into small fragments of 1-3 mm3 in a cell culture dish using surgical scissors or scalpels.

5.      Digest the tissue fragments with 10mL of Tumor Tissue Digestion Solution (K601003) in a 15mL conical tube at 37°C, with variable incubation times ranging from 30 min to 1.5 h. Carefully monitor the digestion process, mixing the content of the tube every 5-10 min by shaking vigorously or pipetting the mixture up and down. The digestion process could be finished when most of tissue fragments are able to pass through the 1mL pipette tips.

6.      Add FBS to the tissue digestion mixture to a final concentration of 2%, and filter using a 100 μm cell strainer.

7.      Collect and centrifuge the filtered cells at 250g for 3 min at 4 °C. In case of a visible red pellet, aspirate the supernatant, and resuspend the pellet using 2mL of Red Blood Cell Lysis Solution (E238010) to lyse the erythrocytes at room temperature for 1 min and centrifuge at 250g for 3 min at 4°C.

8.      Aspirate the supernatant and resuspend the pellet in Cancer Organoid Basal Medium, centrifuge at 250g for 3 min at 4°C, repeat this step once more time.

9.      Aspirate the supernatant and resuspend the pellet in Matrigel. The Matrigel should be kept on ice to prevent it from solidifying. Perform the process as quickly as possible. The amount of Matrigel used depends on the size of the pellet. Approximately 10,000 cells should be plated in 25 μL of Matrigel.

CRITICAL: Do not overly dilute the Matrigel (Matrigel should be >70% (Matrigel vol/Total vol)), as this may inhibit the proper formation of the solid droplets.

10.   Plate the Matrigel containing organoids at the bottom of 24-well cell culture plates in droplets of ~30 μL each around the center of the well..

CRITICAL: Once the organoids are resuspended in Matrigel, proceed with plating as quickly as possible, as the Matrigel may solidify in the tube or pipette tip. Do not let the Matrigel touch the wall of wells.

11.   Place the culture plate into a humidified incubator at 37 °C and 5% (vol/vol) CO2 for 15-25 min to let the Matrigel solidify.

12.   Prepare the required amount of breast cancer organoid complete medium.

13.   Once the Matrigel droplets have solidified (15-25 min), open the plate and carefully add 500 μL of organoid complete medium to each well.

CRITICAL: Do not add the medium directly on top of the Matrigel droplets, as this might disrupt the droplets.

14.   Place the culture plate in a humidified incubator at 37 °C and 5% (vol/vol) CO2.

15.   Change the medium every 3-4 d by carefully aspirating the medium from the wells and replacing it with a fresh, pre-warmed organoid complete medium.

16.   Closely monitor the organoid formation. Ideally, patient-derived breast cancer organoids should be passaged for the first time between 7 and 10 d after the initial plating.

Splitting and Passaging of Organoids

17.   Pipette up and down to disrupt the Matrigel and transfer the organoid suspension to a 1.5 mL conical tube.

18.   Pipette the organoid suspension up and down to mix thoroughly by pipetting against the bottom of the tube to create pressure, which will aid the removal of Matrigel.

19.   Centrifuge organoids at 250g for 3 min at room temperature.

20.   Aspirate the supernatant and split organoids using either Organoid Dissociation Solution (E238001) or by mechanical disruption. For Organoid Dissociation Solution-based cell dissociation, resuspend the pellet in 0.2 mL of Organoid Dissociation Solution, pipette up and down and incubate at 37 °C until organoids fall apart. Pipette up and down with a filter tip for ≥8 times every 2 min to aid in the disruption of the organoids. Closely monitor the digestion to keep the incubation time in the Organoid Dissociation Solution to a minimum. In case of mechanical disruption, resuspend the pellet in 1.5 mL of Cancer Organoid Basal Medium. Carefully pipette the organoid suspension up and down 30 times by pipetting against the bottom of the tube to create pressure, which will aid organoid disruption.

CRITICAL: Do not dissociate in Organoid Dissociation Solution for >7 min, as this may result in poor organoid outgrowth or even loss of the culture. As a rule of thumb, digestion is complete if a mixture of small lumps of cells (consisting of 10–50 cells) can be observed.

21.   After shearing is complete, wash once by topping up with 1 mL of Cancer Organoid Basal Medium, and centrifuge at 250g for 3 min at room temperature.

22.   Aspirate the supernatant and resuspend the organoid pellet in 70% (vol/vol) Matrigel, and plate organoids in droplets at the bottom of a culture plate as described in Steps 10. After plating is complete, transfer the plate into a humidified incubator at 37 °C and 5% (vol/vol) CO2 for 15–25 min.

23.   Pre-warm breast cancer organoid complete medium at 37 °C.

24.   After the Matrigel droplets have solidified (15–25 min), carefully pipette pre-warmed medium into the wells.

25.   Place culture plates in a humidified incubator at 37 °C and 5% (vol/vol) CO2 until the organoids are needed for further experiments.