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Generating and maintaining uniform 3D spheroids in long term cultures

  • R&D Team 1
  • 1 - PHC Corporation of North America (PHCNA)

Aug 30, 2021


This instruction explains the basic operating procedure for generating and maintaining longterm three-dimensional spheroid cultures on PrimeSurface® 96 Slit-well plates. Instructions recommended here are the best operating protocols but depending upon your cell line and assay, some optimizations may be required. 


Enhanced stem cell culturing in regenerative medicine cell culturing involves frequent media replacement to provide nutrition to growing cells. In a standard 96 well ultra-low cell attachment plate, media aspiration or dispensing has to be done extremely carefully to avoid disturbing the unattached spheroid, making this a time consuming operation. PrimeSurface 96 Slit-well Plate allows media handling and exchange for 96 well plates efficiently with one step dispensing or aspiration for all 96 wells, decreasing the pipetting time by over 80% while minimizing the risk of spheroid damage. The organoids grow more comparably sized in the slit well plate compared with either 10cm dishes or traditional 96 well plates. The interconnectedness of the wells ensures more biological consistency of the different wells. The benefit is the much faster time to feed cultures. Feeding also only requires a pipette while feeding traditional wells requires multichannel pipettes and media boats. The slit well plates reduce time of handling and cost, particularly for longterm cultures. The new design of ultra-low attachment 3D plates facilitate easy handling of media exchange without disrupting spheroid formation.

Benefits of slit-well plates for spheroid culture

Reagents and Equipment

PrimeSurface® 96 Slit-well plates.



Procedures of cell spheroid formation 

  1. Open the packaging of a new Slit-well Plate under the hood.
  2. Seed cells in the plate at a density of 6,000-9,000 cells/100 μL/well.
  3. The recommended volume of the above cell  suspension is approximately 100 μL.
  4. As the maximum capacity of each well is 100 μL, do not exceed 100 μL volume for cell suspension per well.
  5. The formed spheroids are apt to slip through  the slit or protrude outside the well if the  number of seeded cells is insufficient.
  6. Dispense the cell suspension at the bottom of each well (section B). (Do not dispense it in  section a of the well, i.e. in the slit section).
  7. Culture the cells in the incubator.
  8. After every 24-48 hrs, check for spheroid formation.
  9. Gently and carefully handle the plate during this first 24-48 hrs, as the spheroids being formed in the initial stage are fragile and can break apart.
  10. Dispense fresh culture media (20 mL per plate) at the corner of the plate using a 25 mL pipette.
  11. The total volume of the dispensed culture media is 30 mL per plate, i.e. an aliquot of 10 mL during cell seeding, plus 20 mL in this step (as stated above).
  12. The maximum capacity of the plate is 40 mL/plate.
  13. Dispense media slowly (it should take about 20 seconds to dispense 20 mL volume to the plate).
  14. Gently shake the plate back and forth in all directions a couple of times to ensure that all the wells have been evenly covered with media.
  15. Place the plate in the incubator.

Procedure for exchanging culture media 

  1. Take the plate out of the incubator and check the spheroid formation under a microscope before exchanging the culture media. 
  2. Aspirate the media from a corner of the plate with a 25 mL pipette and dispose of it.
  3. Apply the pipette tip to the corner of the plate and gently aspirate the old culture media while slightly tilting the plate.
  4. The plate must be tilted in order to properly aspirate the culture media.
  5. The recommended aspirating volume is 15 to 20 mL (takes about 20 seconds).
  6. Dispense fresh media from the corner of the plate with a 25 mL pipette. This volume should be the same as that of aspiration step (2). 
  7. Note that the capacity of the slit plate is 40 mL/ plate. It is advised to stay with total volume of 30 mL/plate. 
  8. Dispense media slowly (takes 20 sec) to avoid any overflow. 
  9. Gently shake the plate back and forth in all directions a couple of times to ensure that all the wells and corners of the plate have been evenly covered with media.

Time Taken

Long-term cultures

Notes and Comments

  1. The product and instruction are subject to change. 
  2. This product is for research purpose only.
  3. PrimeSurface® 96 Slit-well Plate, is designed to form and maintain uniform spheroid for long term cultures. 
  4. New and innovative design of plate with slits on the upper half of each well.
  5. Media aspirating and dispensing is efficiently performed from the corners of the plate while keeping the spheroid formation undisturbed in wells.


Not available


Gurmeet Kaur Jan 24, 2022

I am very interested in your plates and protocol. Can someone get in touch with me to discuss your protocol and also information on staining the spheroids in the wells with PD markers. I would like to receive few plates to try before bulk order. Thank you. My contact information is as follows: Gurmeet Kaur, M.S. Molecular Pharmacology Branch Division of Cancer Treatment and Diagnosis, NCI Frederick National Laboratory for Cancer Research (FNLCR) 1050 Boyles Street Bldg. 321 / Room 122 Frederick MD 21702-1201 Office: (301) 846-1994 Lab: (301) 846-1932 Fax: (301) 846-7443 E:

Marshall Kadin Jan 24, 2022

Interesting approach. Has it been used for lymphoma cultures? What is the cost of plates? Thanks, MEK

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