We can understand the heaviness in your heart when seeing this title, especially when dealing with difficult-to-culture organoids. After painstaking efforts to establish a culture, the organoids fail to proliferate post-passage. Today, let's analyze the reasons for organoid passaging failure from the following three aspects.

 

I. Organoid Passaging Techniques

 

The most critical factor affecting organoid passaging is the passaging technique. Some delicate organoids are like greenhouse flowers, highly susceptible to various "storms." For instance, matrix gel cold conditions (temperature and duration), centrifugation conditions (temperature, speed, and duration), loss of organoids during supernatant removal, digestion conditions (temperature and duration), and pipetting force can all pose significant challenges akin to the " eighty-one tribulations" in the journey to the West.

 

(1) Matrix Gel Cold Temperature and Duration

 

When passaging organoids, collect the matrix gel and organoid suspension from 5-10 wells (using a 24-well plate as an example) into a 15 mL centrifuge tube using Organoid Passaging Buffer (abs9730). Adjust the volume to 14 mL with the buffer. Then, cold the matrix gel using methods such as 4℃ for 30 minutes, 0℃ for 30 minutes, or -20℃ for 5 minutes. While most organoids tolerate these conditions, some are overly sensitive to temperature or duration. For temperature-sensitive organoids, it is advisable to use 4℃ for 30 minutes or 0℃ for 30 minutes; for those sensitive to duration, -20℃ for 5 minutes is recommended.

 

(2) Centrifugation Temperature, Speed, and Duration

 

Centrifugation may cause damage to organoids, primarily including:

A. Mechanical Stress Damage: The mechanical stress generated during centrifugation can disrupt organoid cells. This damage may involve cell membrane rupture, internal structural damage, and loss of cell function.

B. Temperature Effects: Centrifugation temperature significantly impacts organoid preservation. High temperatures can固化 the matrix gel, affecting organoid separation and storage. Therefore, centrifugation at 4℃ is recommended to prevent heat damage.

After matrix gel solidification, when performing density gradient centrifugation, a horizontal rotor centrifuge is suggested. Control the speed at 200g-300g, set the temperature at 4℃, and keep the duration within 5 minutes.

 

(3) Loss of Organoids During Supernatant Removal

 

Stratification Phenomenon (Buffer, Matrix Gel, Organoid Sediment)

 

After centrifugation, organoids typically stratify into three layers: buffer, matrix gel, and organoid sediment. Normally, we retain the organoid sediment and discard the upper buffer and matrix gel layers. However, if the number of primary cultured organoids is low, the organoid sediment layer will also be minimal. In such cases, we must not only preserve the organoid sediment but also retain the lower 2/3 or even the entire matrix gel layer to minimize organoid loss (organoids may remain in the matrix gel layer). When dispensing new gel, ensure its volume is at least 1.5 times that of the residual gel to guarantee the adhesiveness of the matrix gel.

 

 

Sometimes, after centrifugation, organoids may form two layers: buffer and a matrix gel-organoid suspension. In such cases, it is advisable to retain the lower 2/3 or even the entire matrix gel-organoid suspension layer to minimize organoid loss (organoids may remain in the matrix gel layer). When dispensing new gel, ensure its volume is at least 1.5 times that of the residual gel to guarantee the adhesiveness of the matrix gel.

 

(4) Digestion Temperature and Duration

 

When digesting and passaging the centrifuged organoid suspension, particular attention must be paid to controlling digestion temperature and duration. Excessive digestion can damage organoids in two ways:

A. Cell Membrane Damage and Cell Death: Over-digestion may disrupt cell membranes, releasing cell contents into the culture medium and causing cell death.

B. Increased Apoptosis: Prolonged trypsin digestion may increase the apoptosis rate. Excessive digestion can damage cell membranes and affect the functionality and integrity of cell surface receptor proteins.

 

Although digestive enzymes exhibit optimal activity at 37℃, excessive digestive power is not necessarily beneficial for organoids. Typically, organoid digestion can be performed in a biosafety cabinet at room temperature, allowing better control of digestion extent and timely termination based on microscopic examination. It is recommended to limit digestion time to 2-3 minutes (the longer the digestion time, the greater the potential enzyme-induced damage to organoids).

 

(5) Pipetting Force

 

Pipetting in organoid culture primarily generates two types of forces: mechanical damage and shear stress.

 

A. Mechanical Damage: This refers to cell damage caused by pipette tip scraping or cell-to-cell collisions. To minimize this damage, reduce pipetting force to just enough to detach and disperse cells. Contact or grinding of the pipette tip with the cell layer can lead to direct cell death. Therefore, control the distance between the pipette tip and the culture dish bottom to avoid direct contact.

B. Shear Stress: Shear stress refers to the phenomenon where the cross-section of an object experiences relative movement along the direction of an external force. In cell culture medium, if there are many bubbles, cells located between bubbles and the medium may be subjected to strong shear stress when bubbles burst, causing cell fragmentation.

 

If mechanical pipetting is used for passaging, the force and number of pipetting actions significantly impact organoid viability. When using a 1 mL pipette tip, operate gently to avoid shear stress-induced organoid injury. A sterile Pasteur pipette can be used instead of a pipette tip to reduce harm. Limit pipetting to within 30 times and observe under a microscope. Once organoids detach from the matrix gel and form cell clusters, stop pipetting. Therefore, when pipetting organoids, it is crucial to control the force and technique to minimize cell damage.

 

II. The Organoids Themselves

 

Lung Cancer Organoids Show Little Growth within One Week Post-Passage

 

(1) Organoid Activity Level

 

Different types of organoids vary significantly in activity. Those with high activity, such as endometrial cancer and colorectal cancer organoids, are relatively easy to culture and can be passaged up to over 30 times. However, some organoids have inherently low activity. For example, thyroid cancer organoids can only be maintained in primary culture and fail to proliferate post-passage. Additionally, liver cancer and prostate cancer organoids are also notoriously difficult to culture.

 

(2) Organoid Quantity

 

If the number of organoids in primary culture is low, for instance, in a 24-well plate with 50 μL of matrix gel-cell cluster suspension per well, and fewer than 20 organoids develop, it is typically a case of low organoid quantity. In such situations, improper passaging techniques may lead to significant organoid loss. The resulting low cell cluster density, coupled with insufficient paracrine signaling regulation, makes organoid proliferation difficult, causing the "failure to grow" phenomenon. Thus, the number of primary organoids directly impacts successful passaging.

 

III. Nutrient System

 

(1) Culture Medium

 

Complete culture medium (with added factors) is recommended to be stored in aliquots at -20℃ for up to one year; when stored at 4℃, it is advisable to use it within two weeks, as factor activity gradually declines after one month.

 

Storing culture medium at 4℃ for an extended period (over one month) can reduce its efficacy. How does this affect organoid passaging?

For example: the same bottle of culture medium is used for digestion and passaging. Primary culture thrives, but passaged cultures fail. When inferring the cause, one might initially rule out the culture medium, as using the same bottle for both primary and passaged cultures would imply that if the medium were problematic, the primary culture would also fail. However, the primary culture succeeds while the passaged culture does not. There is an overlooked factor here. It is known that organoids typically exhibit reduced proliferation after multiple passages (e.g., decreased number and size), making further passaging impossible. This is because adult stem cells or cancer stem cells in organoid cultures gradually lose their stemness (self-renewal and differentiation capabilities) with each passage. In primary organoid cultures, these stem cells exhibit strong stemness and can proliferate even with culture medium stored at 4℃ for an extended period. However, as passages continue, stemness diminishes, increasing the demand for factor activity in the culture medium. Using medium stored at 4℃ for an extended period will then make it difficult to expand organoids.

 

(2) Organoid Passaging Digestion Medium

 

The impact of digestion medium on organoid passaging is reflected in two aspects:

A. Whether the passaging digestion medium is appropriate; improper enzymes can damage organoids.

B. Passaging digestion medium is recommended to be stored in aliquots at -20℃ for up to one year; when stored at 4℃, it is advisable to use it within two weeks, as enzyme activity gradually declines after one month.

Firstly, the digestive power of enzymes depends on the type of enzyme. We have attempted to use trypsin for organoid digestion and found that it causes significant damage to organoids and is not suitable for passaging. Therefore, it is recommended to use a specialized organoid passaging digestion medium (abs9520), which employs multiple mild digestive enzymes for gentler organoid digestion.

Secondly, if the digestion medium is stored at 4℃ for an extended period, enzyme activity gradually decreases, prolonging the digestion time required for organoids. Improperly extending digestion time can lead to over-digestion and compromise organoid viability.

 

(3) Matrix Gel

 

Matrix gel is recommended to be stored in aliquots at -20℃ for up to one year; when stored at 4℃, it is advisable to use it within two weeks, as its adhesiveness and factor activity gradually decline after one month.

 

Matrix gel serves two functions: it acts as a scaffold to maintain the ellipsoid shape of organoids and contains a variety of factors that promote organoid growth. When matrix gel is stored at 4℃ for an extended period, both its adhesiveness and factor activity are significantly reduced. Under these conditions, the matrix gel-organoid dome structure is prone to detachment from the culture dish bottom and floating, and it also fails to meet the proliferative demands of organoids.

 

Recommended Organoid Culture Products

Catalog No.	Product Name	Specifications
abs9495	Matrix Gel (Low Factor, Phenol Red-Free)	1.5mL×4/1.5mL×8
abs9445	Organotial Human Colorectal Cancer Organoid Culture Kit	1kit
abs9786	Organotial Human Intrahepatic Cholangiocarcinoma Organoid Culture Kit	1kit

 

 

 

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