Table 1.

Advantages and disadvantages of different methods of NK cell expansion for adoptive transfer studies.

Source of NK cells	Activation or stimulation	Advantages	Disadvantages			Refs
Ex vivo Expanded
Umbilical cord		Higher percentage of NK cells than other sources	Relatively low number of NK cells		(11, 12)
blood		Contains NK progenitors that can differentiate into NK cells, and that are absent from	Potentially lower cytotoxicity that PB derived NK cells
		Have higher expression of bone marrow homing receptors
iPSC		Can be generated in an off-the-shelf manner	Requires specialized expertise to generate NK cells from iPSC and to maintain clinical grade iPSC cell lines		(13, 14)
		iPSCs can be propagated indefinitely and can generate NK cells for multiple treatments	Relatively low level in the peripheral blood after infusion
		Generate purer NK cell populations with known KIR haplotypes
Peripheral blood (CD3 depletion, CD56 enrichment) CD3/CD19 depleted	IL-2 stimulation	Enhanced viability after cryopreservation, enhanced cytotoxicity	Short-term treatment (12-16 hours) does not result in sufficient NK cell expansion on its own		(15, 16)
	IL-15 stimulation	Enhanced NK cell cytotoxicity	Can result in functional exhaustion of NK cells if stimulation is too long
	IL-2 + IL-21 stimulation	IL-21 enhances NK cell cytotoxicity, cytokine secretion, and ADCC	IL-21 may trigger NK cell apoptosis
	IL-12 + IL-15 + IL-18 stimulation	Generates memory-like NK cells
		Increases expression of CD25 on NK cells, making them more amenable to IL-2 mediated expansion
	IL-15 + IL-21 stimulation	Method can be combined with CD34 selection to concurrently purify stem cells for infusion	Contain other contaminating cells such as monocytes and dendritic cells		(17, 18)
CD34+ selection followed by differentiation to NK cells		Generates high purity of NK cells	Greater variability and poorer yields of differentiated NK cells than from umbilical cord blood		(19)
		Can generate more CD34+ cells than umbilical cord blood
Feeder cell lines
	Irradiated autologous PBMCs		Limited availability as the feeder cells must be obtained from a pretreated patient	(7, 8, 14, 20–23)
			Generally poorer expansion than from allogeneic donor PBMC
	Irradiated K562 with membrane bound IL-15/4-1BB/IL-2	Enhanced NK cell cytotoxicity	Increased incidence of aGVHD after infusion into patients in one trial
		Efficient ADCC
	Irradiated K562 with membrane bound 4-1BB/IL-21	Increased telomere length	Allogeneic feeder cell lines must pass stringent quality control measures in order to be acceptable for infusion into patients
		Enhanced persistence
	Plasma membrane particles of K562 with membrane bound IL-21/4-1BB/IL-2	Lower risk of transmission of feeder culture contaminant to patient	NK cells expanded with plasma membrane particles tended to have a lower expression of the bone marrow homing molecule CD62L compared to feeder culture cell line expansion
		Similar cytotoxicity profile to those expanded with feeder culture
	EBV transformed lymphoblastoid cells	Enhanced NK cell cytotoxicity
		Increased NK cell cytokine secretion
	Priming cell lines	Primed NK cells are KIR independent and do not require cytokine stimulation		(15, 24)
	CTV-1 leukemia cell line
Biomaterials
	Hyaluronic acid-based biodegradable polymeric scaffold					(25)
In vivo Expanded
CIML NK cells	expanded with low dose IL-2	Massive in vivo expansion	Specialized processing facility with high manhours required			(21, 26, 27)
		Enhanced cytotoxicity
NK	IL-2	Expansion of NK cells	May also expand regulatory T-cell populations			(28)
			Associated with capillary leak syndrome, constitutional symptoms and cardiac toxicity
NK	IL-15	Expansion and persistence of NK cells	The subcutaneous form is associated with neurologic toxicity and cytokine release syndrome			(28)