IPS-SpheresTM

Enables high cell yields for hPSCs expansion

$150.00$2,200.00
  • Quantity : In 150 mg or 300 mg vials.
  • Shelf life : 1 Year
  • Store at : 4 oC

IPS-SpheresTM

$150.00$2,200.00

Enables high cell yields for hPSCs expansion

Clear

Overview

IPS-SpheresTM microcarriers (MCs) provide a foundation for the scalable and robust production of human pluripotent stem cell (hPSCs)-derived functional cells in large numbers, by means of an integrated propagation and differentiation bioprocess in a defined environment [1]. The MCs can be used for generating uniform EBs-like cell aggregates under agitation cultures and subsequently directly differentiated towards various lineages or functional cells (e.g. cardiomyocytes [2,3,4] and neural progenitor cells [5]). The advantages of this approach are: high cell yields; scalable; controlled aggregate size; negligible labor-intensive manual intervention allowing expansion and differentiation of hPSCs in one vessel unit [1,2].

Applications include:

– Enables high cell yields for hPSCs expansion

– Can be used for directed differentiation to any lineages or functional cells with different medium, such as cardiomyocytes and neuroprogenitors

– Alternative to EBs formation, giving higher cell yields and easier handling than “cutting colonies” or Aggrewells

This product is for research use only and is not for use in diagnostic procedures.

Protocols

Additional Reagents Required

  1. hPSCs growth media (e.g. mTeSRTM 1 and StemFIT)
  2. Cell dissociation reagent (e.g. TrypLETM Express and AccutaseTM)
  3. Phosphate Buffered Saline, Ca2+ Mg2+ free (PBS)
  4. 6-well Ultra-low adhesion (ULA) culture plates (e.g. CorningTM Catalog number: 3471)
  5. Erlenmeyer shake flask, 125mL with Vent cap (e.g. Corning® Catalog number: 431143)
  6. Orbital shaker (e.g. Biosan PSU-10i)
  7. 40 mm cell strainers (e.g. FalconTM Blue cell strainers, Catalog number: 352340)

General Protocol

Section I. Bead Preparation

  1. Aseptically pipette the entire contents of a vial of IPS-SpheresTM MC suspension to a sterile 15-mL tube.
  2. Rinse vial twice with 1 mL of sterile PBS and add rinse solution to the 15-mL tube containing MC suspension.
  3. Once the MC have settled, the PBS may be aseptically removed by careful aspiration or pipetting. Care should be taken not to aspirate the MC.
  4. Reconstitute the desired amount of MC in media. Recommendation: 20 mg MC for 5 mL culture in 6-well ULA plate.

Section II. Cell Seeding

  1. Preparation of a single cell suspension of undifferentiated hPSCs according to your own cell dissociation protocol. Recommendation: Gently dissociate the hPSCs by using TrypLETM Express or AccutaseTM is recommended.
  2. Add the appropriate volume of undifferentiated cells (1x106 cells) to the 6-well ULA plate containing MC. Thus, final cell culture contains 2x105 cells/mL and 4 mg/mL MC.
  3. After adding cells, immediately pipette gently 3 times with 10-mL serological pipette to allow evenly distribution of cells throughout the culture vessel.
  4. Agitate (orbital shaking: 110 rpm) the plate for 2 hours to allow cell attachment to MC. (Figure 1 shows the image of cells attached on IPS-SpheresTM MCs after 2 hours agitation cultures).
  5. After 2 hours, place the plate in a 37oC incubator with 5% CO2 and 95% humidity and culture for 7 days under static or agitated conditions (orbital shaking: 110 rpm).
  6. Replenish 80% of the culture spent medium every day. When exchanging culture medium, allow cell-MC aggregates to settle 5 minutes, and using a serological pipette, slowly remove 80% of culture spent medium, and replace with equal volume of fresh hPSCs growth medium without causing much disturbance to the cell-MC aggregates. (Figures 2 and 3 show the image of cell-MC aggregates after 7 days of cultivation under static and agitated conditions, respectively).

 

Microcarrier 1

Figure 1 shows the phase-contrast microscopy image of hESC HES-3 cells attached on the IPS-Spheres after 2 hours post-seeding under agitation. Scale bar: 500mm.

 

Microcarrier 2

Figure 2 shows the phase-contrast microscopy image of cell-MC aggregates after 7 days of cell expansion on IPS-SpheresTM under static condition. Scale bar: 500mm.

 

 

Section III. Passaging hPSCs from MC to MC

  1. Break up 6-7 day’s cultured cell-MC aggregates using 1-mL filtered pipet tip by continuously aspirating and dispensing until an even distribution of cell-MC clumps is achieved (~200-300 mm in size).
  2. Measure the total volume of medium in each well of 6-well plate using a 5-mL serological pipette. For accurate reporting on cell yield at the end of a passage, it is important to take note of the actual culture volume to calculate the total number of cells.
  3. Take two samples of 200 µL each to determine the viable cell concentration of cell clumps using the NucleoCounter® NC-300. In brief, the cell sample is added with an equal volume of the lysis buffer Reagent A, which disrupts the plasma membrane of the cells and exposes the nuclei for staining (acridine orange and DAPI), provided by the manufacturer. The stabilizing Reagent B is then added into the sample in an equal volume of as Reagent A. The final cell sample mixture is loaded into the slide at ~10 µl. The instrument reads the numbers of nuclei and provides the cell count. Instead of using the NucleoCounter, other standard nuclear count methods can be used. For example, cell-MC clumps can be dissociated into single cells using dissociating enzymes (TrypLETM Express and AccutaseTM) and viable cells quantified using the trypan-blue-exclusion method. Note: No filtration of MC is needed.
  4. Seed desired amount (2x105 cells/mL) of the cell-MC aggregates into a new vessel with newly prepared MC (prepare desired amount of MC in media as aforementioned, Section I, #4).
  5. Agitate the plate for 2 hours to allow cell attachment to MC.
  6. After 2 hours, place the plate in a 37oC incubator with 5% CO2 and 95% humidity and culture for 7 days under static condition.
  7. Feed the cells every day by removing 80% of the spent medium and adding equal volume of fresh hPSCs growth medium.

Section IV. Expansion of hPSCs using Shake flask

  1. Prepare 80 mg MC in 20 mL for a 125-mL Erlenmeyer Shake flask, as described in Section I.
  2. Transfer 4x106 viable cells on MC (i.e. 2x105 cells/mL), cultured previously in Section II or Section III, into the Shake flask.
  3. After adding cells, immediately pipette gently 3 times with 10-mL serological pipette to allow evenly distribution of cells throughout the culture vessel.
  4. Incubate the culture on the orbital shaking platform for 7 days inside a 37oC incubator with 5% CO2 and 95% humidity and set the speed to 100-110 rpm.
  5. Feed the cells every day by removing 80% of the spent medium and adding equal volume of fresh hPSCs growth medium. Briefly, allow cell-MC aggregates to settle 5 minutes, and using a 10-mL serological pipette, slowly remove spent medium without causing much disturbance to the cell-MC aggregates.

 

Microcarrier 3

Figure 3 shows the phase-contrast microscopy image of cell/IPS-SpheresTM aggregates after 7 days in 6-well ULA plate or shaker flask expansion under agitation. Scale bar: 500mm.

 

Section V. Cell harvesting

  1. Separate media from cell-MC aggregates by centrifugation.
  2. Wash cell-MC aggregates 1-2 times with PBS.
  3. Add appropriate volume of cell dissociation reagent (e.g. TrypLETM Express and AccutaseTM) to cell-MC aggregates. Pipetting up and down and incubation at 37oC is required to aid in cell detachment.
  4. Detached cells can be separated from the MC by filtering the cells through a 40 mm cell strainer. Multiple PBS rinses may be required to ensure all cells are collected.

Download Detailed Protocol

Videos

Publications

1. Sivalingam J et al. 'Superior Red Blood Cell Generation from Human Pluripotent Stem Cells Through a Novel Microcarrier-Based Embryoid Body Platform'. Tissue Eng Part C Methods. 2016 Aug;22(8):765-80.


2. Lam AT et al. ‘Integrated processes for expansion and differentiation of human pluripotent stem cells in suspended microcarriers cultures’ Biochem Biophys Res Commun. 2015 Sep 15.


3. Lam AT et al. ‘Conjoint propagation and differentiation of human embryonic stem cells to cardiomyocytes in a defined microcarrier spinner culture’. Stem Cell Res Ther. 2014 Sep 15;5(5):110.


4. Lam AT et al. ‘Improved Human Pluripotent Stem Cell Attachment and Spreading on Xeno-Free Laminin-521-Coated Microcarriers Results in Efficient Growth in Agitated Cultures’. Biores Open Access. 2015 Apr 1;4(1):242-57.


5. Ting S et al. ‘An intermittent rocking platform for integrated expansion and differentiation of human pluripotent stem cells to cardiomyocytes in suspended microcarrier cultures’. Stem Cell Res. 2014 Sep;13(2):202-13.


6. Brady J et al. ‘Microcarrier suspension cultures for high-density expansion and differentiation of human pluripotent stem cells to neural progenitor cells’. Tissue Eng Part C Methods. 2013 Feb;19(2):166-80.