HCM-BROD-0009-C25

1. Check all containers for leakage or breakage.
2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.

To prepare the complete medium for this organoid model, please refer to the Organoid Media Formulation #3.

ATCC offers the recombinant proteins, small molecules, and other supplements to make this complete medium; Organoid Growth Kit 1B (ATCC ACS-7101) provides these supplements in a convenient, pre-portioned, ready-to-reconstitute format that does not require aliquoting or storage once prepared.

Seeding density: We recommend seeding this model at 0.25 - 1 x 10⁶/ viable cells in 100 µL of ECM per well of a 6-well plate.

ECM: We recommend culturing this model in ATCC Cell Basement Membrane (ATCC ACS-3035) or Corning Matrigel. Include 10 µM ROCK Inhibitor Y-27632 (ATCC ACS-3030) in medium for the first 2-3 days following subculture.

For a brief overview of the thawing procedure see our quickstart guide Thawing Cryopreserved Human Organoids.

Initiating culture from frozen vials: For a brief overview of the thawing procedure see our quickstart guide Thawing Cryopreserved Human Organoids.

Seeding density: 0.25 - 1 x 10⁶/ viable cells in 100 µL of ECM per well of a 6-well plate. Alternatively, split at 1:2-1:4 every 7-10 days. For example, collect organoids from 100 µL of extracellular matrix (ECM) from a single well of a 6-well plate and re-seed into 2-4 wells of a 6-well plate in 100 µL ECM per well.

Media renewal: Perform a complete medium change every 2-3 days. Include 10 µM ROCK Inhibitor Y-27632 (ATCC ACS-3030) in medium for the first 2-3 days following subculture.

For a brief overview of the subculture and expansion of organoids see our quickstart guide Subculture and Expansion of Human Organoids Protocol.

For more details on the handling and culture of organoids see our methods paper in Current Protocols in Cell Biology.

For a brief overview of the cryopreservation procedure for organoids see our quickstart guide Organoid Cryopreservation Protocol.

For more details on the handling and culture of organoids see our methods paper in Current Protocols in Cell Biology.

The product is provided 'AS IS' and the viability of ATCC^® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

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