Shrimp Cell 3D Culture via Matrigel Spheroids
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Solution Overview
Problem
Establishing immortalized shrimp cell lines is hindered by the difficulty in culturing shrimp cells, which are sensitive to proteases and fail to divide in existing 2D culture systems, limiting research on viral diseases and vaccine production in shrimp aquaculture.
Innovation Solution
A method for 3D shrimp cell culture using matrigel with a complete medium supplemented with fetal bovine serum and shrimp ovarian extract, along with specific conditions for CO2 and temperature, allows for the growth and passage of shrimp cells without enzymatic digestion, promoting stereoscopic growth and survival.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If 2D culture methods are used for shrimp cells, then cells can be cultured in traditional flasks and plates, but shrimp cells fail to divide and are extremely sensitive to proteases including trypsin
Solution Approach 1:
The patent transitions from traditional 2D culture to 3D spheroid culture by embedding shrimp cells in matrigel droplets that form spherical structures. This dimensional change enables cells to aggregate and divide properly, overcoming the failure of 2D cultures to support shrimp cell proliferation.
Solution Approach 2:
Matrigel serves as an intermediary substrate that provides the necessary extracellular matrix environment for shrimp cells to adhere, aggregate, and divide. The matrigel droplets act as a protective medium that eliminates the need for protease treatment during passaging.
2Ease of operation
If proteases like trypsin are used for cell passage, then cells can be detached from culture surfaces, but shrimp cells are extremely sensitive and suffer harm
Solution Approach 1:
Matrigel serves as an intermediary substrate that provides the necessary extracellular matrix environment for shrimp cells to adhere, aggregate, and divide. The matrigel droplets act as a protective medium that eliminates the need for protease treatment during passaging.
Solution Approach 2:
The patent changes the physical state and composition of the culture system by using matrigel-based spheroids instead of adherent monolayers. This parameter change allows cells to be passaged by mechanical disruption of the spheroid structure rather than enzymatic digestion, avoiding protease damage.
3Object-affected harmful factors
If 3D culture is attempted for shrimp cells, then proteases may not be needed for passage, but current efforts on 3D culture and passage of shrimp cells are poor
Solution Approach 1:
The patent transitions from traditional 2D culture to 3D spheroid culture by embedding shrimp cells in matrigel droplets that form spherical structures. This dimensional change enables cells to aggregate and divide properly, overcoming the failure of 2D cultures to support shrimp cell proliferation.
Solution Approach 2:
The patent employs a dynamic passaging process where spheroids are formed, allowed to grow, then disrupted and redistributed to new matrigel droplets. This dynamic cycle of formation-growth-disruption-redistribution enables continuous culture without protease treatment.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The method enables continuous culture and passage of shrimp cells with improved survival and division rates, laying the foundation for establishing immortalized shrimp cell lines and enhancing in vitro growth abilities.
Implementation Method 1
adding a shrimp cell suspension to culture wells with matrigel
Implementation Method 2
adding a cell recovery solution with the same osmotic pressure as the complete medium for shrimp cells
Implementation Method 3
centrifuging the dissolved matrigel-cell suspension for 5 to 10 min at 3,000×g at 4° C.
Implementation Method 4
The culture is performed in a 3% CO2 incubator at 28° C.
Data Source
AI summary
By establishing effective methods for shrimp 3D cell culture and passage, the present invention provides a technology of continuous shrimp cell culture intended for the establishment of immortalized shrimp cell lines. The present invention provides a preparation method of matrigel for 3D cell culture of shrimp by optimizing an additive proportion of matrigel. The present invention further provides a technology of separation and 3D cell culture of shrimp haemolymph cells, where shrimp haemolymph cells adhere to and grow on the surface of the matrigel in the form of a single round cell and a cell pellet/cellular spheroid, with survival and growth abilities being superior to 2D culture effects. The above technology is achieved by optimizing a formula of complete medium for shrimp cells, selecting the medium as an anticoagulant and a diluent for shrimp haemolymph cells, selecting a 3D culture method for surface-adhered growth in the matrigel.