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Cell separation using cryogel-based affinity chromatography



In cell affinity chromatography, type-specific cell separation is based on the interaction between cell-surface receptors and an immobilized ligand on a stationary matrix. This protocol describes the preparation of monolithic polyacrylamide and polydimethylacrylamide cryogel affinity matrices that can be used as a generic type-specific cell separation approach. The supermacroporous monolithic cryogel has highly interconnected large pores (up to 100 μm) for convective migration of large particles such as mammalian cells. In this protocol, they are functionalized to immobilize a protein A ligand by a two-step derivatization of epoxy-containing cryogel monolith (reaction with ethylenediamine and glutaraldehyde). Target cells were labeled with specific antibodies and then they were captured in the cryogel through affinity with protein A. These specifically captured cells were recovered in high yields while retaining their viability by mechanical squeezing of the spongy and elastic cryogel matrices. The suggested cell separation protocol takes <30 min for complete separation on a preprepared protein A–immobilized cryogel column.

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Figure 1: Cryogelation process: polymer or monomer is mixed in aqueous solvent and then the whole system is incubated at subzero temperature for cryopolymerization and/or gelation, along with ice crystal formation (ice crystals function as a porogen).
Figure 2
Figure 3: The cell separation strategy used for stem cell separation from umbilical cord blood.
Figure 4: Mechanical squeezing of the cryogel matrix for the recovery of bound cells.
Figure 5: Physical characteristics of the monolithic cryogel chromatography column.
Figure 6: Scanning electron micrograph of the inner part of the supermacroporous cryogel-protein A matrix loaded with lymphocytes.
Figure 7: The cell separation efficiency on an affinity-based cryogel chromatographic column (per milliliter adsorbent).


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We acknowledge the financial support from the Department of Biotechnology (DBT) and the Department of Science and Technology (DST), Ministry of Science and Technology, Government of India.

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The protocol was jointly conceived and written by both the authors.

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Correspondence to Ashok Kumar.

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Kumar, A., Srivastava, A. Cell separation using cryogel-based affinity chromatography. Nat Protoc 5, 1737–1747 (2010).

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