標(biāo)簽歸檔：organoid

bioGenous類(lèi)器官基質(zhì)膠 Organoid Culture ECM (Reduced Growth Factor)(M315066)

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類(lèi)器官基質(zhì)膠 Organoid Culture ECM (Reduced Growth  Factor)

貨號(hào)：M315066

規(guī)格：10mL

品牌：bioGenous

產(chǎn)品介紹

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.

技術(shù)參數(shù)

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類(lèi)器官傳代消化液 Organoid Dissociation Solution（E238001）

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Organoid Dissociation Solution 類(lèi)器官傳代消化液

貨號(hào)：E238001

規(guī)格：100ml

500ml

品牌：bioGenous

產(chǎn)品介紹

bioGenousTM Organoid Dissociation Solution（類(lèi)器官傳代消化液）可應(yīng)用于多種哺乳動(dòng)物（如人、鼠、豬、蝙蝠、牛等）來(lái)源類(lèi)器官的常規(guī)傳代，可使類(lèi)器官?gòu)幕|(zhì)膠中分離，并可使其溫和地消化為小細(xì)胞簇或單細(xì)胞，同時(shí)保持其傳代后的生長(zhǎng)活力。

技術(shù)參數(shù)

產(chǎn)品信息

產(chǎn)品組成

貨號(hào)

規(guī)格

儲(chǔ)存條件及周期

bioGenousTM Organoid Dissociation Solution

E238001

100mL/500mL

2-8°C，12 個(gè)月

類(lèi)器官消化過(guò)程中可能應(yīng)用到的其他試劑

試劑

推薦貨號(hào)

 Organoid Basal Medium

B213152 & B213151

Fetal Bovine Serum, FBS

–

類(lèi)器官的消化傳代

1.         向回收后的類(lèi)器官中加入 5-10倍類(lèi)器官基質(zhì)膠混合物體積的Organoid Dissociation Solution（類(lèi)器官傳代消化液），吹打混勻后在37°C條件下孵育1-8分鐘使類(lèi)器官解離（需提前取所需體積的消化液在37℃條件下預(yù)熱，通常單層結(jié)構(gòu)類(lèi)器官的消化時(shí)間為0.5-3分鐘，多層或者體積較大的類(lèi)器官消化時(shí)間為3-8分鐘）。

注意：在此操作過(guò)程中須仔細(xì)監(jiān)測(cè)消化過(guò)程，避免過(guò)度消化。在消化過(guò)程中，可使用移液器吹打混勻幫助消化。也可實(shí)時(shí)取少量消化懸液于顯微鏡下觀(guān)察消化情況，當(dāng)觀(guān)察到較多的單細(xì)胞或直徑在50μm以下的細(xì)胞簇后，即可認(rèn)為消化完成。

2.         在確認(rèn)消化完成的懸液中，加入至少五倍體積的類(lèi)器官基礎(chǔ)培養(yǎng)基進(jìn)行稀釋?zhuān)瑥亩K止消化作用。

注意：時(shí)間較長(zhǎng)的消化過(guò)程結(jié)束后可適當(dāng)加入胎牛血清（Fetal Bovine Serum, FBS）至終濃度2%-5%以保證消化后細(xì)胞的活力。

3.         將上步驟所獲類(lèi)器官懸液進(jìn)行離心（水平離心轉(zhuǎn)子，150-300g，3分鐘），棄上清，再次加入基礎(chǔ)培養(yǎng)基重懸類(lèi)器官沉淀。

4.         將上步驟所獲類(lèi)器官懸液進(jìn)行離心（水平離心轉(zhuǎn)子，150-300g，3分鐘），棄上清后所獲類(lèi)器官可用于后續(xù)類(lèi)器官培養(yǎng)、凍存等實(shí)驗(yàn)操作。

bioGenous類(lèi)器官凍存液 Organoid Cryopreservation Medium（Serum Free）（E238023）

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Organoid Cryopreservation Medium（Serum Free）  類(lèi)器官凍存液

貨號(hào)：E238023

規(guī)格：100ml

品牌：bioGenous

產(chǎn)品介紹

bioGenousTM Organoid Cryopreservation Medium （類(lèi)器官凍存液）可應(yīng)用于多種哺乳動(dòng)物（如人、鼠、豬、蝙蝠、牛等）來(lái)源的類(lèi)器官和細(xì)胞系的低溫長(zhǎng)期保存，該凍存液不含血清，不含任何動(dòng)物來(lái)源成分，含有 10% DMSO。

技術(shù)參數(shù)

產(chǎn)品信息

產(chǎn)品組成

貨號(hào)

規(guī)格

儲(chǔ)存條件及周期

bioGenousTM Organoid Cryopreservation Medium（Serum Free）

E238023

100mL

4℃，3年

類(lèi)器官（或細(xì)胞）的凍存和復(fù)蘇

需選用生長(zhǎng)狀態(tài)良好的類(lèi)器官（或細(xì)胞）進(jìn)行凍存實(shí)驗(yàn)。 

類(lèi)器官（或細(xì)胞）的凍存 

1.       向離心后的類(lèi)器官（或細(xì)胞）中加入預(yù)冷的（2-8℃）Organoid Cryopreservation Medium（類(lèi)器官凍存液），吹打混勻后迅速轉(zhuǎn)移至細(xì)胞凍存管中（凍存管內(nèi)細(xì)胞懸液體積應(yīng)大于0.5mL）。

注意：每1mL凍存液推薦凍存1×103-1×107個(gè)細(xì)胞或?qū)?yīng)細(xì)胞量的類(lèi)器官。

2.       將凍存管放入細(xì)胞凍存程序降溫盒內(nèi)（降溫盒須提前平衡至室溫或4℃），隨后立即將降溫盒放入-80 ℃超低溫冰箱中；也可將凍存管進(jìn)行人工梯度降溫處理，如4℃ 靜置10分鐘，-20℃保存1小時(shí)，-80℃保存過(guò)夜。

3.       次日或12小時(shí)后將凍存管于低溫條件下（不高于-70℃，推薦使用干冰或原降溫盒）轉(zhuǎn)移至液氮中長(zhǎng)期保存。 

類(lèi)器官（或細(xì)胞）的復(fù)蘇

 4.       提前在37℃條件下預(yù)熱類(lèi)器官（或細(xì)胞）所需的基礎(chǔ)培養(yǎng)基。

5.       在37℃的水浴中快速解凍細(xì)胞凍存管，當(dāng)凍存管內(nèi)凍存物僅剩些許冰渣殘留時(shí)立即停止水浴并及時(shí)轉(zhuǎn)移至潔凈操作臺(tái)。

6.       將凍存懸液轉(zhuǎn)移至離心管中，緩緩加入5-10倍體積的預(yù)熱的基礎(chǔ)培養(yǎng)基，輕輕混勻。

7.       將上步驟所獲類(lèi)器官（或細(xì)胞）懸液進(jìn)行離心（水平離心轉(zhuǎn)子，150-300g，3分鐘），棄上清，再次加入基礎(chǔ)培養(yǎng)基重懸類(lèi)器官（或細(xì)胞）沉淀。

8. 將上步驟所獲類(lèi)器官（或細(xì)胞）懸液進(jìn)行離心（水平離心轉(zhuǎn)子，150-300g，3分鐘），棄上清后所獲類(lèi)器官（或細(xì)胞）可用于后續(xù)類(lèi)器官（或細(xì)胞）的培養(yǎng)。

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

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肺腺癌 Lung Adenocarcinoma Organoid Kit

貨號(hào)：K2138-LA

規(guī)格：100ml

500ml

品牌：bioGenous

產(chǎn)品介紹

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.

技術(shù)參數(shù)

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肝內(nèi)膽管癌 Cholangiocarcinoma Organoid Kit（K2104-LB）

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肝內(nèi)膽管癌 Cholangiocarcinoma Organoid Kit

貨號(hào)：K2104-LB

規(guī)格：100ml

500ml

品牌：bioGenous

產(chǎn)品介紹

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.

技術(shù)參數(shù)

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肝實(shí)質(zhì)細(xì)胞癌 Hepatocellular Carcinoma Organoid Kit（K2105-HCC）

發(fā)表于由

肝實(shí)質(zhì)細(xì)胞癌 Hepatocellular Carcinoma Organoid Kit

貨號(hào)：K2105-HCC

規(guī)格：100ml

500ml

品牌：bioGenous

產(chǎn)品介紹

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.

技術(shù)參數(shù)

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宮頸癌 Cervical Cancer Organoid Kit（K2169-CC）

發(fā)表于由

宮頸癌 Cervical Cancer Organoid Kit

貨號(hào)：K2169-CC

規(guī)格：100ml

500ml

品牌：bioGenous

產(chǎn)品介紹

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.

技術(shù)參數(shù)

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結(jié)直腸癌 Colorectal Cancer Organoid Kit（K2103-CR）

發(fā)表于由

結(jié)直腸癌 Colorectal Cancer Organoid Kit

貨號(hào)：K2103-CR

規(guī)格：100ml

500ml

品牌：bioGenous

產(chǎn)品介紹

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.

技術(shù)參數(shù)

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）

發(fā)表于由

卵巢癌 Ovarian Cancer Organoid Kit

貨號(hào)：K2168-OC

規(guī)格：100ml

500ml

品牌：bioGenous

產(chǎn)品介紹

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.

技術(shù)參數(shù)

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）

發(fā)表于由

乳腺癌 Breast Cancer Organoid Kit

貨號(hào)：K2147-BC

規(guī)格：100ml

500ml

品牌：bioGenous

產(chǎn)品介紹

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.

技術(shù)參數(shù)

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.