cryopreserved dendritic cells for intratumoral immunotherapy do not require re-culture prior to...

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Journal of Immunological Me

Research paper

Cryopreserved dendritic cells for intratumoral

immunotherapy do not require re-culture

prior to human vaccination

Justin Johna,b,T, Angus Dalgleisha, Alan Melcherc, Hardev Pandhaa

aDepartment of Oncology, St. George’s Hospital Medical School, Cranmer Terrace, London SW17 ORE, United KingdombBioVex Ltd, Milton Park, Abingdon, Oxfordshire, United Kingdon

cDepartment of Clinical Oncology, St. James Hospital, University of Leeds, Leeds, United Kingdom

Received 20 August 2004; received in revised form 16 November 2004; accepted 20 December 2004

Available online 28 January 2005

Abstract

Dendritic cell (DC) immunotherapy for cancer has shown great promise so far. The ability to deliver dendritic cells directly

into tumours where they are capable of acquiring tumour antigens prior to stimulating specific T cell responses has been

demonstrated both in animal models and human patients. Clinical grade DCs can be grown from peripheral blood monocytes in

the absence of foetal calf serum (FCS) and cryopreserved to generate plentiful identical aliquots thus avoiding repeated

venesection. However, the approach is still limited by the necessity to return thawed DCs to culture prior to injection. It would

be more advantageous to directly inject the DCs whilst still in the freezing medium and thus prevent the need for further

manipulation. Whilst several reports have shown that cryopreserved DCs can survive for over 72 h when returned to culture,

there is no information regarding the longevity of cells maintained in the freezing medium after thawing. In this report we have

shown that DCs may remain in freezing medium for up to 1 h without affecting their survival, phenotype or function. This

period of time is sufficient to allow for any delays incurred between the preparation of the DCs and time taken to be

administered within a standard clinical setting.

This study demonstrates that clinical grade DCs can be cryopreserved and thawed whilst retaining the ability to acquire

exogenous antigenic material required for intratumoural immunotherapy. The survival of these cells within the freezing medium

without the requirement for re-culture expands their availability for administration directly to the tumours of patients in non-

specialist centres that do not have the appropriate facilities for DC re-culture.

D 2005 Elsevier B.V. All rights reserved.

Keywords: Dendritic cells; Immunotherapy; Cryopreservation

0022-1759/$ - s

doi:10.1016/j.jim

T Correspondi

Kingdom. Tel.:

E-mail addr

thods 299 (2005) 37–46

ee front matter D 2005 Elsevier B.V. All rights reserved.

.2004.12.014

ng author. Department of Oncology, St George’s Hospital Medical School, Cranmer Terrace, London SW17 ORE, United

+44 20 87251255x0809; fax: +44 20 87250158.

ess: [email protected] (J. John).

J. John et al. / Journal of Immunological Methods 299 (2005) 37–4638

1. Introduction

Dendritic cells (DC) are the most potent antigen

presenting cells known with the capacity to capture,

process and present antigens in order to stimulate

specific T cells (Steinman, 1991). The recognition of

their proficiency has been utilised and harnessed for the

development of DC-based immunotherapy for cancer.

Current clinical trials have relied on the generation of

DC from either peripheral blood monocytes or CD34+

stem cells (Nestle et al., 1998; Mackensen et al., 1999;

Holtl et al., 2002). Repeated venesection or leukapha-

resis required to generate sufficient numbers of either

cell type is undesirable for patients who are often

already debilitated from their disease.

The use of ex vivo differentiated DCs for intra-

tumoural injections has resulted in increased survival

and decreased tumour size in both animal experiments

and human trials (Melero et al., 1999; Murakami et

al., 2004; Triozzi et al., 2000) The success of this

route of administration relies upon sufficient in situ

uptake of antigens and the subsequent stimulation of

specific CTL-mediated immune responses (Melero et

al., 1999).

Current data suggests that it is possible to generate,

modify or antigen load, cryopreserve in aliquots and

revive sufficient numbers of clinical grade DCs for

sequential vaccination thereby avoiding the need for

repeated venesection (Feuerstein et al., 2000; John et

al., 2003). This approach is itself currently limited by

the necessity to return thawed DC to culture prior to

vaccination (Feuerstein et al., 2000; Pecher et al.,

2001; Schuler-Thurner et al., 2002). Removing the

need for re-culture of thawed cells would be a simple,

but extremely important, step forward in the DC

vaccine approach. Not only would it simplify the

overall strategy, but it would also reduce the risks of

microbial contamination and allow cryopreserved

vaccines to be shipped to non-specialist centres for

administration after thawing at the bedside.

In this study, we have investigated whether

immature DCs can be successfully cryopreserved

and recovered without further manipulation. It would

be of great advantage if DCs displayed an ability to

remain in freezing medium over periods of time,

without compromising their activity and thus enabling

medical staff to thaw vaccines at the bedside prior to

administration.

2. Materials and methods

2.1. Isolation of peripheral blood mononuclear cells

Peripheral blood (50 ml) was obtained from

healthy individuals by venous puncture and collected

in sodium heparin vacutainers. The blood was diluted

1:1 with Hanks balanced salt solution (HBSS).

Peripheral blood mononuclear cells (PBMCs) were

isolated by Ficoll density gradient centrifugation.

Mononuclear cells were collected from the interface,

washed once in HBSS before being re-suspended in

the appropriate assay medium.

2.2. Generation of research-grade monocyte-derived

dendritic cells

PBMCs were resuspended in growth medium

(RPMI-1640 containing 10% heat-inactivated FCS

(both purchased from Invitrogen, Paisley, UK), 50 U/

ml penicillin and 50 Ag/ml streptomycin (both

purchased from Sigma-Aldrich, Poole, UK) at a final

concentration of 3�106 cells/ml. Cells were incubated

in 100 mm plastic Petri dishes for 2 h at 37 8C. Non-adherent cells were removed by vigorous pipetting

and the remaining adherent cells cultured in growth

medium supplemented with 100 ng/ml research grade

recombinant human GM-CSF (Peprotech EC Ltd,

London, UK) and 50 ng/ml recombinant human

interleukin-4 (Peprotech) at 37 8C 5% CO2. Research

grade DCs were generated by day 7 of culture.

2.3. Generation of clinical-grade monocyte-derived

dendritic cells

For the generation of clinical grade DC, PBMCs

were isolated as outlined above and monocytes

separated using CD14 MicroBeads (MiltenyiBiotec,

Surrey, UK) following the manufacturer’s instruc-

tions. Purified monocytes were cultured at 106/ml in

RPMI medium containing 10% heat-inactivated

pooled human AB serum (Cambrex, Wokingham,

UK), 50 U/ml penicillin, 50 Ag/ml streptomycin, 100

ng/ml clinical grade recombinant human GM-CSF

(Immunex, Seattle, WA) and 50 ng/ml GMP grade

recombinant human interleukin-4 (CellGenix, Frei-

burg, Germany). Clinical grade DCs were obtained by

day 7 of culture.

J. John et al. / Journal of Immunological Methods 299 (2005) 37–46 39

2.4. Direct surface staining for immunophenotyping

Cells were harvested and washed in wash buffer

(PBS containing 0.5% BSA and 0.1% sodium azide,

all purchased from Sigma). Aliquots of 105 cells were

labeled with the relevant fluorochrome-conjugated

antibody for 30 min on ice in the dark. Cells were also

incubated with an irrelevant isotype-matched control

antibody to compensate for non-specific binding. The

cells were washed in wash buffer and the cell pellet

fixed with 200 Al CellFix (BDIS, Oxford, UK).

Samples were analysed within 24 h (stored at 4 8Cin the dark) on a FACScan flow cytometer (BDIS);

routinely 10,000 events were collected. Dead cells and

debris were gated out on the basis of their light scatter

properties. The antibodies used were CD14 (clone

TUK4); CD80 (clone MEM-233); CD83 (clone

HB15e); CD86 (clone BU63); HLA-DP,DQ,DR

(clone WR18) and HLA-A,B,C (clone W6/32) and

isotype controls all obtained from Serotec, Oxford,

UK.

2.5. Cryopreservation of dendritic cells

Dendritic cells were counted and the pellet chilled

on ice for 5 min. The cells were resuspended in

freezing medium (Table 1) at a final concentration of

3�106cell/ml and aliquoted to cryovials. The cryo-

vials were placed in freezing containers (Nalgene,

Rochester, USA) containing 2-isopropanol and then

transferred to a �70 8C freezer where the rate of

cooling was controlled to �1 8C/min (manufacturer’s

information). After 72 h the cryovials were transferred

directly to vapour-phase liquid nitrogen for long-term

storage.

Frozen DCs were recovered from storage in liquid

nitrogen by thawing in a 37 8C water bath over 100 s.

The cells were washed in 15 ml of warm growth

medium before being resuspended in an appropriate

medium for experimentation.

Table 1

Components of freezing medium used to cryopreserve human

dendritic cells for either (A) research or (B) clinical trials

Mix DMSO% RPMI% HS% FCS%

A 12 44 – 44

B 12 44 44 –

2.6. DC recovery and survival in vitro

Cryopreserved DCs were defrosted as outlined

above and either assessed immediately or allowed to

remain in the freezing medium for various lengths of

time. At each time point the DCs were washed in 15

ml warm growth medium before being resuspended at

5�105/ml in growth medium supplemented with 100

ng/ml recombinant human GM-CSF and 50 ng/ml

recombinant human interleukin-4 and returned to

culture. At various time points, aliquots of cells were

removed and assessed for viability by trypan blue

exclusion or the number of viable, apoptotic and

necrotic cells using an APO-BrdU in situ apoptosis

detection kit (BDIS) following the manufacturer’s

instructions.

Apoptosis was confirmed by detecting the trans-

location of membrane phosphatidylserine in the

presence of membrane integrity. DCs were washed

in ice-cold PBS and resuspended at 106/ml in binding

buffer (10 mM HEPES/NaOH pH 7.4, 140 mM NaCl,

2.5 mM CaCl2). 100 Al of cells were aliquoted to

tubes containing 5 Al Annexin V-FITC (BDIS) and 10

Al PI (Sigma) and incubated for 15 min at room

temperature in the dark. 400 Al of binding buffer were

added and samples analysed within 1 h by flow

cytometry.

2.7. Antigen acquisition

This method was adapted from that published

elsewhere (Sallusto et al., 1995). 500 Al of DC

(3�105/ml) in growth medium were incubated at 4

8C (background) or 37 8C for 10 min to equilibrate the

temperature. Dextran–FITC (40,000 MW Sigma) or

Lucifer yellow (Molecular Probes, Paisley, UK) were

added at 1 mg/ml to each sample and incubated for a

further 60 min at the same temperature. Washing the

cells twice in ice-cold PBS quenched the endocytic

activity and removed any free dextran–FITC and LY.

Cells were fixed in 200 Al of CellFix and routinely

5000 cells were analysed by flow cytometry.

2.8. Mixed lymphocyte reaction (MLR)

The stimulatory function of the DCs was assessed

by their ability to induce proliferation in allogeneic

non-adherent PBMCs in vitro. Graded numbers of


DCs resuspended in assay medium (RPMI 1640

containing 10% heat inactivated pooled human serum

50 U/ml penicillin and 50 Ag/ml streptomycin) were

incubated with 106 allogeneic non-adherent PBMCs

resuspended in the same medium. Proliferation was

measured on day 5 following 18 h of pulsing with 1

ACi [3H]thymidine (Amersham Biosciences, Bucks,

UK) per well. Mean values of triplicates were

measured and expressed as counts per minute (cpm).

Stimulation indices (S.I.) were calculated by dividing

the test sample cpm by that of the background cpm(T

cell alone+DC alone).

2.9. Antigen-specific T cell proliferation

Fresh DCs were loaded with 10 Ag/ml tetanus

toxoid (Calbiochem, Nottingham, UK) for 2 h at 37

8C under serum-free conditions. The cells were then

washed twice and frozen as above. Revived DC were

washed once in HBSS. Freeze-thawed cells were

recovered from freezing medium at various time

points in assay medium (RPMI 1640 containing

10% heat-inactivated pooled human serum, 50 U/ml

penicillin and 50 Ag/ml streptomycin). Autologous

responder cells consisted of non-adherent PBMCs

collected at the time of initial DC preparation and

frozen for 6 days. These cells were revived and

allowed to recover in assay medium for 24 h. Freeze-

thawed DCs and responder cells were counted and

resuspended in assay medium at 105 cells/ml and 106

cells/ml, respectively.

2.10. Antigen-specific ELISpot

The IFNg ELISpot assay followed the manufac-

turer’s instructions (Mabtech, Nacka, Sweden).

Briefly, Silent Screen 96-well plates with biodyne

membranes (Nalgene, Life Technologies, USA) were

coated with the primary capture antibody (clone 1-

D1K) overnight at 4 8C. Plates were washed once withPBS to remove non-bound antibody before the

addition of PBS/10% AB serum for 2 h at room

temperature to block non-specific binding. The plates

were washed once with PBS before adding 100 Al ofDC and 100 Al of autologous non-adherent PBMCs in

assay medium to each well (ratio 1:10). The cells were

incubated for 24 h at 37 8C in a 5% CO2 humid

incubator. Cells were removed by washing six times

with PBS–Tween 20 (PBS–T20) and the wells

incubated with 1 Ag/ml biotinylated detection anti-

body (clone 7-B6-1-biotin) for 3 h at room temper-

ature. Plates were washed six times with PBS–T20

before incubation with streptavidin–alkaline phospha-

tase (1:1000 in PBS/0.5% FCS) for 2 h at room

temperature. Following six final washes with PBS–

T20, the plate was incubated with alkaline phospha-

tase conjugate substrate as recommended by manu-

facturer (Bio-Rad, Herts, UK) for up to 30 min until

spots appeared. Washing the plate in tap water and

then allowing it to dry in air stopped the reaction. Spot

forming colonies (SFC) were enumerated using the

Zeiss Axioplan 2 ELISpot counter (Image Associates,

Oxford, UK).

2.11. Statistical analysis

Data are expressed as meanFS.D. Statistical

significance was determined using a paired two-tailed

Student’s t-test. A P value of b0.05 was considered as

significant.

3. Results

3.1. Generation and cryopreservation of research and

clinical grade DCs

To date, the cryopreservation of dendritic cells has

been achieved by optimising both the freezing

medium and the rate of cooling (Feuerstein et al.,

2000; John et al., 2003; Pecher et al., 2001). However,

the parameters for thawing and their fate prior to

injection have not been as thoroughly investigated,

especially if the DCs were intended to be directly

available in the clinic or at the bedside. While DCs

generated in FCS are acceptable for research pro-

cesses, they are inappropriate for clinical trials. DCs

generated in the presence of human serum (HS) are

phenotypically more mature than those grown in FCS

as shown by the increase in expression of class I, class

II, CD86 and CD83 surface molecules (Fig. 1). The

DCs can be cryopreserved and thawed successfully

with minimal effect on viability, irrelevant of the

source of serum (FCS or HS) used in the freezing

medium. In order to minimise the requirement for

additional sample manipulation prior to vaccination

Fig. 1. Research (FCS) and clinical grade (HS) DC surface marker

expression. The phenotype of day 7 DCs grown in the presence of

FCS or HS was characterised with specific antibodies and analysed

by flow cytometry. Panels show staining for the indicated molecules

(filled) compared to appropriate isotype control (empty).


the process would greatly benefit from direct injection

of DCs in the freezing medium. However, the period

between defrosting and administration can differ and

as such may compromise the efficacy of the cells prior

to inoculation. The viability of thawed DCs is

maintained even when the cells remain in the freezing

medium at room temperature for up to 2 h (Fig. 2).

This suggests that within this time period the freezing

0

2040

6080

100

Fresh 0

Tim

% V

iabl

e

Fresh

Fig. 2. Viability of thawed DCs at various time points in freezing medium

freezing medium containing either FCS or HS. Following at least 72 h in th

at various times at room temperature in the freezing medium. On recovery

freezing medium before being washed and viability assessed by trypan bl

medium is not toxic to the DCs even at this high

concentration of DMSO (12%).

3.2. Viability and survival of thawed DCs

The effect on their long-term survival of maintain-

ing DCs in freezing medium was evaluated by

returning the thawed cells to culture and assessing

the induction of apoptosis as assessed by the

incorporation of BrdU. There was no significant

difference (PN0.05) in the number of apoptotic DCs

detected in clinical grade DC samples returned

immediately to culture or after recovery from cry-

opreservation and maintained in the freezing medium

for up to 1 h. The increase in the percentage of

apoptotic cells after 72 h and 120 h in culture was

comparable in all DC samples independent of the

length of time they had remained in freezing medium

(Fig. 3). Annexin V and propidium iodide staining

confirmed the similarity in the percentage of apoptotic

clinical grade DCs either returned immediately to

culture or following maintenance in freezing medium

for up to 1 h after thawing (Fig. 4). The survival of

these cells in culture at 72 h followed an identical

pattern, as did the percentage of apoptotic and

necrotic cells, for all time points (Table 2).

3.3. Antigen acquisition of cryopreserved clinical

grade DCs

The functional activity of thawed clinical grade

DCs after cryopreservation was assessed in order to

determine whether the cells would be capable of

orchestrating an immune response. Initially, the DCs

30 60 120

e (min)

FCS HS

. DCs generated in medium containing FCS were cryopreserved in

e vapour phase of liquid nitrogen, they were thawed and maintained

DCs were either used immediately (0 h) or allowed to remain in the

ue dye exclusion (n=4).

Table 2

Survival of clinical grade DCs after 72 h reculture is identical for

each time point of cells maintained in freezing medium

Time

(min)

% of population

Viable Apoptotic Necrotic

Fresh 90.6 2.4 7

0 0 64.9 5.7 29.4

24 44.36 24.36 31.28

48 52.79 36.16 11.05

72 43.25 27.85 28.9

30 0 63.58 4.58 31.84

24 42.98 22.74 53.36

48 40.24 33.87 25.89

72 43.25 25.65 31.1

60 0 73.12 4.58 22.3

24 47.82 19.54 32.64

48 45.28 27.84 26.88

72 45.98 27.14 26.88

Clinical grade DCs cryopreserved in freezing medium containing

HS were thawed and maintained at various times at room temper-

ature in the freezing medium. The DCs were washed and returned to

culture in medium without exogenous cytokines. DCs were assessed

for the percentage of viable (PI�/AnnV�), early apoptotic (PI�AnnV+) and necrotic (PI+/AnnV+) cells by flow cytometry. Data

are representative of three independent experiments.

0

10

20

30

40

50

60

70

0 72 120

Time (h)

% B

rdU

+ve

0 30 60

Fig. 3. Apoptosis of DCs after thawing and re-culture for up to 5

days. Cryopreserved DCs were thawed and maintained for various

times at room temperature in the freezing medium (0 min, 30 min or

60 min). The DCs were washed and returned to culture in medium

without exogenous cytokines. At various times during re-culture,

the DCs were assessed for the percentage of apoptotic cells by BrdU

incorporation as assessed by flow cytometry (n=4).


would be need to acquire antigenic material from the

injected tumour in order to process and present to the

relevant T cells. DCs incubated at 37 8C with Lucifer

yellow accumulated this surrogate marker by fluid-

phase endocytosis to a level greater than that at 4 8C(quiescent cells). Comparable results were obtained

with the receptor-mediated uptake of FITC–dextran

highlighting the fact that these DCs have retained

multiple mechanisms for antigen uptake (Fig. 5).

Similar results were observed with cryopreserved

research grade DCs (data not shown).

0

20

40

60

80

100

120

Fresh 0 24 48 72

Time (h)

% V

iabl

e (P

I-/A

nnV

-)

0 30 60

Fig. 4. Viability of thawed clinical grade DCs during reculture for up to 72 h. DCs cryopreserved in freezing medium containing HS were

thawed and maintained at various times at room temperature in the freezing medium (0 min, 30 min or 60 min). The DCs were washed and

returned to culture in medium without exogenous cytokines. Each day of culture DCs were assessed for the percentage of viable cells by

propidium iodide (PI) and Annexin V (AnnV) flow cytometry (n=4).

/

3.4. Stimulatory capacity of cryopreserved DCs

The ability of thawed DCs to stimulate T cells

was measured to determine whether this activity was

compromised if not tested immediately. DCs recov-

ered from freezing medium at different time points

post thawing were co-cultured with allogeneic T

0123456789

20000 10000 5000 2500

DC / well

S.I.

T 0 30 60 Fresh

**

*

Fig. 7. DCs maintain their ability to stimulate allogeneic T cells afte

5 days of reculture. Clinical grade DCs cryopreserved in freezing

medium containing HS were thawed and maintained at various

times at room temperature in the freezing medium (T0—0 min

30—30 min; 60—60 min). The DCs were then returned to culture

for 72 h before being washed and co-cultured with allogeneic T cells

in a MLR (data representative of three independent experiments)

TPb0.005, TTPb0001.

A

Lucifer Yellow FITC-Dextran

B

Fig. 5. Endocytosis of surrogate markers by clinical grade DCs post

cryopreservation. Thawed DCs were incubated at either 4 8C(empty) or 37 8C (filled) in the presence of 1 mg/ml Lucifer yellow

(A) or FITC–dextran (B) for 1 h prior to analysis by flow cytometry.


cells in a standard MLR. DCs cultured and frozen

in medium containing FCS stimulated allogeneic T

cells to proliferate to a greater extent compared to

DCs cultured in HS at the highest ratio of DC/T

cell. However, there were no significant differences

between fresh DCs and thawed DCs maintained in

either freezing medium for up to 1 h (Fig. 6).

Clinical grade DCs maintain their ability to stim-

ulate proliferation even when returned to culture for

5 days after being maintained in the freezing

medium for up to 1 h after thawing although this

level is less than that induced by fresh DCs (Fig. 7).

The ability of cryopreserved DCs to stimulate a

tetanus toxoid (TT) antigen-specific recall response

was evaluated by both an autologous T cell

proliferation assay and ELISpot. DCs grown and

cryopreserved in medium containing HS induced

proliferation of TT-specific T cells to a similar

A B

DC / well

S.I.

S.I.

20000 10000 5000 2500 20000

4035302520151050

4035302520151050

10000 5000 2500

DC / well

Fig. 6. Thawed research and clinical grade DCs retain ability to stimulate allogeneic T cells after recovery. DCs cultured and frozen in medium

containing either FCS (A) or HS (B) were thawed and maintained at various times (o fresh, 5 0, n 30, E 60 min after thawing) at room

temperature in the freezing medium. The DCs were washed and co-cultured with allogeneic T cells in a MLR (n=4). There is no significan

difference between DCs maintained in freezing medium for any time point ( PN0.05).

r

;

.

degree as FCS DC. Both fresh and HS DCs induce

less antigen-specific proliferation when compared to

their FCS DC counterparts (Fig. 8A). However, all

3 DC samples induced similar levels of IFNg

secretion TT-cells (Fig. 8B).

4. Discussion

The use of cryopreserved antigen loaded or

genetically modified DCs has already facilitated a

number of clinical trials of cancer immunotherapy.

The advantages of this approach over repeated

venesection for fresh DCs are obvious in terms of

t

50

Fresh FCS AB

40

30

20

10

00 30

Time (min)

Spot

s/10

6 PB

MC

s

60

200

AB FCS Fresh

150

100

A

B

**

50

020000

S.I.

10000 5000

DC/well

2500 0

Fig. 8. Antigen-specific T cell stimulation maintained after

cryopreservation and retention in freezing medium. Tetanus toxoid

pulsed DCs cryopreserved in freezing medium containing either

FCS or HS were thawed and maintained at various times at room

temperature in the freezing medium. The DCs were washed and co-

cultured with autologous T cells (n=4). (A) Proliferation or (B)

IFNg ELISpot data are meansFS.D. of triplicate samples.

(TPN0.05).


safety, feasibility, cost and reproducibility. Moreover,

it permits a high degree of dquality controlT prior toinjection of the patient. Avoiding the need to re-

culture thawed DC vaccines to aid their recovery

would be a further important step forward. Vaccines

may then be prepared centrally and patients treated in

non-specialist centres, as no cell culture facility would

be required. The elimination of further processing post

cryopreservation would also reduce the possibility of

contamination.

We have shown that DCs generated in HS are more

mature than those generated in FCS with respect to the

repertoire of surface molecules they express. The

increase in expression of class I and II molecules as

well as CD86 would permit the increased stimulation

of specific T cells in vivo following vaccination. DCs

may be cyropreserved in medium containing either

FCS or HS and successfully recovered. Assessments

of FCS DCs after thawing over short periods of time

indicate no significant reduction in cell viability or

survival. Furthermore, the thawed DCs retained their

phenotypic expression (data not shown, see John et

al., 2003), morphology, allostimulatory activity and

the ability to generate a recall response to tetanus

toxoid using two assays. Clinical grade DCs frozen in

medium containing HS maintained their ability to

stimulate both allogeneic and autologous T cells even

when retained in the freezing medium at room

temperature for up to 1 h. The levels induced by HS

DCs were lower than those induced by FCS DC but

this enhancement in immune responses has been

attributed directly to the presence of FCS and is

unlikely to be due to variations in the DCs themselves

(Mackensen et al., 1999; Dols et al., 2003).

The time periods needed in the clinic where

vaccines would ideally be administered as soon as

possible after thawing are clearly well below those

evaluated in this report. If delays in administration of

the vaccine were to occur following recovery of the

DCs, it has been shown that they can be maintained

for at least 1 h without any deleterious alterations to

either viability or function. Successful utilisation of

this protocol also relies on the constituents of the

freezing medium that would be injected together with

the DCs and are therefore a very important factor for

clinical applications. The use of FCS in the generation

of DCs for vaccination has not been approved and it

must be replaced by human plasma, human serum or

serum-free conditions. To date, we have found that

DCs generated under serum-free conditions resulted

in very low cellular yield (data not shown). However,

GMP-grade human serum (HS) can routinely generate

sufficient numbers of mature DCs to warrant under-

going a clinical trial. The clinical grade freezing

medium is acceptable for human trials as it consists of

pooled human AB serum which is already used

widely in clinical practice, and there is extensive

experience and safety data with administration of stem

cells preserved in high concentrations of DMSO (Stiff

et al., 1987).

For clinical applications several variables still need

to be addressed. These include DC purity, maturation


and serum source for DC generation. These clinical

grade DCs are capable of inducing T cell proliferation

to a similar degree as their fresh counterparts even

after the thawed cells have remained in the freezing

medium at room temperature for at least 1 h. These

clinical grade DCs retained for this period also

maintain the capacity for antigen uptake via fluid

phase macropinocytosis and receptor-mediated endo-

cytosis. The ability of these DCs to sample exogenous

antigenic material indicates that they could potentially

acquire antigens from tumour cells in vivo on

administration. It is not entirely clear whether these

mechanisms are sufficient to initiate effective anti-

tumoural responses after vaccination as they may

possibly be affected by the suppressive nature of the

tumour microenvironment (Gabrilovich et al., 1996;

Triozzi et al., 2000). The category of antigens released

from tumours often depends on the mode of cell death

and can result in differing modulations of DC

response. Treatment of the tumour with chemother-

apeutics (Tong et al., 2001; Tanaka et al., 2002; Shin

et al., 2003) or radiation (Kikuchi et al., 2002; Teitz-

Tennenbaum et al., 2003) has been shown to enhance

DC antigen uptake and induce potent immunity. DCs

engineered to secrete immunostimulatory cytokines

such as IL-12 IL-18 or GM-CSF can also induce very

potent antitumour responses after intratumoural injec-

tion by priming CD8 cytotoxic T cells in draining

lymph nodes (Nishioka et al., 1999; Yamanaka et al.,

2003; Sharma et al., 2003). Therefore, there may be

novel approaches, which would augment the ability of

direct intratumoural injection of clinical grade cry-

opreserved DCs in established tumours.

To date, this study is the first to address the

possibility that cryopreserved DCs can be thawed

and allowed to remain in a freezing medium prior to

immunization of the patient thereby avoiding the

need for further manipulation or re-culture. We have

demonstrated that cells prepared in this way remain

viable and completely functional in freezing medium

for up to 2 h before injection. The components of the

DC growth and freezing media have been in routine

clinical use for some time. We now intend to directly

compare the response of cryopreserved DCs directly

administered into tumours in murine models and

subsequently in patients with cancer. This report

makes a contribution to simplifying strategies for

repetitive vaccination using cryopreserved DC, and

will permit non-specialist centres to undertake

clinical trials using DC-based immunotherapy.

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cryopreserved dendritic cells for intratumoral immunotherapy do not require re-culture prior to...

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