New method to produce hemocomponents for regenerative use from peripheral blood: Integration among platelet growth factors monocytes and stem cells

Transfusion and Apheresis Science 42 (2010) 117–124

Contents lists available at ScienceDirect

Transfusion and Apheresis Science journal homepage: www.elsevier.com/locate/transci

New method to produce hemocomponents for regenerative use from peripheral blood: Integration among platelet growth factors monocytes and stem cells Gaetano Caloprisco a,*, Alessio Borean a,*, Sergio De Angeli b, Giovanni Battista Gaio b, Katia Boito b, Laura Del Pup b, Elisabetta Pavan b, Valentina Casale b, Ivone Varinelli c a b c

Dipartimento di Immunoematologia e Medicina Trasfusionale, Ospedale San Martino, viale Europa 22, 32100 Belluno, Italy Dipartimento di Immunoematologia e Medicina Trasfusionale, Ospedale Ca’ Foncello, Treviso, Italy Haemonetics Italia, Italy

a r t i c l e Keywords: Hemocomponents Regeneration Growth factor Monocyte Stem cells

i n f o

a b s t r a c t Recent studies have shown the importance of monocytes/macrophageses and of CD34+ progenitors in tissue regeneration processes. These cells, obtained generally from bone marrow, are seen in damaged tissue. We have studied a method to collect from the peripheral blood, using a cell separator and without stimulation of the patient/donor, a leukocyte platelet concentrated hemocomponent (CLP) for regenerative use which contains platelets, monocytes/macrophages, fibrinogen and CD34+ cells. We appraised the composition and cell functionality of the final hemocomponent during production and cryoconservation. The results show a positive increase in concentration values, in comparison with the pre-collection, of the cells that were involved in regeneration; i.e. the platelets, monocytes and CD34+ cells. These concentrations were also maintained at an effective level during cryoconservation of the hemocomponent. The CLP also demonstrated positive clonogenic potential in culture, showing that the CD34+ progenitors involved in CFU formation are functional in the fresh and thawed product. In brief we have shown that it is possible to produce, in a simple way, a hemocomponent for regenerative use that is standardized, reliable, and is economically feasible. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Hemocomponents for regenerative use are currently produced for local application in different clinical sectors, and for different types of tissue lesions: bony, cutaneous and tendinous [1–9]. The effectiveness of the regenerative stimulus is induced by the platelet growth factor (GFs) that is contained

* Corresponding authors. Tel.: +39 0437 516297x516274; fax: +39 0437 516567 (G. Caloprisco), tel.: +39 0437 516274; fax: +39 0437 516567 (V. Borean). E-mail addresses: [email protected] (G. Caloprisco), [email protected] (A. Borean). 1473-0502/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.transci.2010.01.003

in these hemocomponents; as such platelet derived growth factor (PDGF), Transforming growth factor a1 (TGF-a1) and Insulin like growth factor I (IGF-I) [10–12] are well demonstrated, but now what remains is to specify the interaction between the injured tissue and hemocomponent cells. Recent scientific evidence has also shown the importance, besides the platelets (PLT), of the resident and circulating monocytes/macrophageses and stem cells in the processes of tissue regeneration and differentiation [13–18], in particular of CD34+ cells and endothelial/ hematopoietic progenitors [19–22]. There are different methods of collecting and producing these hemocomponents [23], however, all of these methods are focused fundamentally on the production of a

118

G. Caloprisco et al. / Transfusion and Apheresis Science 42 (2010) 117–124

platelet gel, which is produced by activation with thrombin, to permit the release of GFs. Because of the above-mentioned considerations, for some years now we have been studying hemocomponents that are capable of integrating the regenerative action of platelet GFs with that of mononuclear cells [6,7]. In this study, we have designed and analyzed a new method to produce, from peripheral blood and without cell manipulation, a leukocyte platelet concentrate (CLP) rich in fibrinogen containing the most important cell elements of regeneration: PLT, monocytes and CD34+ cells. For this purpose we have analyzed and compared the composition, concentration and cell functionality of the CLP during each step of the production procedure. Particular attention has been paid to monocytes, endothelial/hematopoietic progenitors, CD34+ and to their clonogenic potential. The possibility to use these hemocomponents for subsequent applications led us to test the stability of the composition and cell functionality of the cryoconservated CLP. We have shown, that with a cell separator, it is easily possible to achieve a hemocomponent for topical use which is standardized, reliable, has valuable regenerative potential and is a reasonable cost. 2. Materials and methods 2.1. Hemocomponents collection We have performed 45 procedures of multicomponent collection from male donors after acquiring informed consent in accordance with our guidelines [24]. The collection procedure was led entirely to simulate the collection procedure in potential patients. We used a cell separator, the Haemonetics MCS+ (Haemonetics Corp., Braintree, MA, USA), a circuit for the collection of peripheral blood stem cells (Code 971E) and a modified separation protocol. The separation parameters were changed, at the end of the procedure, to obtain two intermediary hemocomponents: plasma and a CLP rich in platelets and mononuclear cells. The modified configuration involves recirculation two consecutive times every three cycles. The beginning of the mononuclear cell collection was fixed at 70% of the total transmittance and the survey of erythrocytes to 12%. The volume of erythrocytes collected for every cycle was fixed to 30 ml, and to 10 ml for every recirculation. The anticoagulant used during collection was ACD in a 1:9 ratio. At the end of the procedure, which lasts approximately an hour, two intermediary hemocomponents, plasma and CLP, are collected in separate bags. 2.2. Production and conservation of the final hemocomponent Our procedure requires that the CLP is enriched in the proteins contained in the insoluble fraction of cryoprecipitate. To produce the cryoprecipitate, the bag of plasma is sterilely connected to a satellite bag with a TSCD welder (Terumo Italy, Segrate, MI, Italy) and frozen by a mechanical freezer (Challeng 250, Angelantoni Industrie, Italy) for 30 min to 80 °C. The plasma bag is then slowly thawed to 4 °C for 18 h to get the cryoprecipitate. After removing all

the plasma into the satellite bag, a fraction of the plasma in the satellite bag is re-infused; the cryoprecipitate adherent on the bag walls is then solubilized with the re-infused plasma at ambient temperature. The CLP is temporarily put back in a mechanical agitator at a temperature of 22 °C for 18 h to maintain the cells in suspension. After 18 h the satellite tube of the CLP bag is removed and a short segment containing about 500 ll of CLP is removed for a cell count and clonogenic tests. Cryoprecipitate and DMSO (Cryo suras DMSO, Wak Chemie Medical GMBH, Steinbach, Germany) are then added to the CLP at 15 and 5%, respectively, of the total volume producing the final mixed hemocomponent (CLP-M) [25]. The cryoprecipitate and the DMSO were added using a device for the administration of intravenous solutions (Aries, Biomedical Devices, MI, Italy), after sterile connection to the CLP bag. These operations were performed in a sterile laminar flow cabinet (Steril VBH compact, MI, Italy). After the addition, the bag of CLP-M was gently shaken for 15 min to mix the constituent and favour DMSO penetration into the cells (from the connected tube to the CLPM bag). From the connection tube, a sample containing about 1 ml of CLP-M is removed and is used to perform the cell counts and to measure the fibrinogen. The CLP-M is then divided into eight parts, using pediatric bags which are sterilely connected (Maco Pharma, Tucoing, France). After labelling, the eight pediatric bags containing the CLP-M fractions are rapidly transferred to a mechanical freezer for 30 min at 80 °C. When frozen, the eight CLPM fractions are immediately transferred into a 80 °C freezer (Polar 530 sv, Angelantoni Industrie, Italia) for long term maintenance. 2.3. Production of activators To achieve the formation of a gel from the CLP-M, we used serum rich in thrombin, capable of activating coagulation and causing platelet degranulation. From every procedure two types of serum rich in thrombin were derived, one from whole blood and the other from cryoprecipitate. From the minibag associated with the apheresis kit, samples of whole blood were collected (4.5 ml) with sterile test tubes containing granules of kaolin (S Monovette, Sarstedt, Numbrect, Germany). After 10 min at 37 °C a clot forms, then the tubes are placed in a centrifuge for 10 min at 3000 rpm to produce the supernatant serum which is rich in thrombin. If not immediately used, the test tubes containing the serum are frozen at 80 °C, to be used later. The cryoprecipitate remaining, after mixing with the CLP, is used to produce serum rich in thrombin. The method requires, in a sterile cabinet, the introduction of calcium gluconate to the cryoprecipitate bag; the calcium gluconate was made into an injectable solution that is 10% equivalent to 446 mEq/l of Ca2+ (Monico, VE, Italy), in a volumetric relationship of 1:5 with the cryoprecipitate. Immediately after the introduction of the calcium gluconate, the contents of the bag are put in test tubes (4.5 ml) containing granules of kaolin. The test tubes are then incubated for 10 min to 37 °C to facilitate the fibrin clot formation. After centrifugation for 10 min at

G. Caloprisco et al. / Transfusion and Apheresis Science 42 (2010) 117–124

3000 rpm the supernatant serum is rich in thrombin. If the test tubes are not used immediately they are to be frozen at 80 °C. If greater sterility is required, a variation of the thrombin extraction (from cryoprecipitate) can be used. For a brief time, the cryoprecipitate bag is connected sterilely with a device for the administration of intravenous solutions, through which, in a sterile cabinet, calcium gluconate is added in the 1:5 relationship. The bag is then incubated for 10 min to 37 °C to facilitate the fibrin clot formation; subsequently the clot is squeezed softly to obtain the supernatant serum rich in thrombin. The serum is taken from the bag only at the moment of activation in the operating room; this variation of serum rich in thrombin, if not used immediately, can be preserved at 80 °C. Activation of the CLP-M forms a fibrin platelet gel that is produced by mixing eight parts of CLP-M, one part of calcium gluconate to 10% and one part of serum rich in thrombin in syringes. After mixing, and before it becomes a gel, the content of the syringe is applied directly on the injured tissue or transferred in a sterile container. In bony regeneration, the fragments of bony tissue taken by the surgeon can be included in the gel. When gelatinization is completed in the sterile container, the gel can then be applied on the lesion. 2.4. Characterization of haemocomponents To check the performance of the collection procedure, the production and conservation of the hemocomponents we had to determine the cell composition and the fibrinogen concentration. In particular the platelet (PLT), the erythrocytes (RBC), total leukocytes (WBC), neutrophil granulocytes (NE), lymphocytes (LY), monocytes (MO), mononuclear total cells (MN) and CD34+ progenitors had to be appraised. Cells are counted, excluding the CD34+, in the donor’s blood before the apheresis (pre-AP), in the CLP, in the fresh CLP-M, in the cryo-preserved CLP-M after three months of maintenance at 80 °C and on the thawed product(CLP-MT). CD34+ progenitors are counted 18 h after the collection in the CLP, and after three months on the CLP-MT. The cells were counted using an automatic cell counter (LH500 Beckman Coulter, CA, USA), while for the count and vitality study of CD34+ progenitors we used a flow cytometer with triple marking (FACSanto Becton Dickinson, Saint Josè, CA, USA). The cryoconserved hemocomponent was tested using two whole quotas after thawing out to 37 °C, for cell counts and clonogenic response. Fibrinogen was measured in the donor plasma and in the supernatant plasma of the CLP-M sample, after centrifugation to 5000 rpm for 10 min. For measuring the fibrinogen dosage an automatic blood coagulation analyzer was used (CA 7000 Sysmex Corp., Wakinohoama Kaigandori, Kobe, Japan). To evaluate and compare the performance of the two types of thrombins we measured the time of clot formation; for this we added the serum rich in thrombin and calcium gluconate to a standard sample of CLP-M (with a fibrinogen concentration of 1123 mg/dl) in the volumetric proportions required for activation A test tube containing

119

800 ll of standard CLP-M was incubated for 1 min at 37 °C in a thermostat (Termomixer Eppendorf, Hamburg, Germany), 100 ll of calcium gluconate and 100 ll of thrombin were then added. The time, in seconds, between the addition of the thrombin and the start of clot formation measures the speed of thrombin activation. 2.5. Clonogenic progenitor assays The clonogenic growth assay (CFU assay) was done according to the method of Miller [26]. To summarize, samples of CLP containing 4.5  105 nucleate cells were put in 3 ml of semisolid ground MethoCult GF H4435 (StemCell Technologies, Vancouver) containing: rh Stem Cell Factor (50 ng/ml), rh Granulocyte Macrophage Colony Stimulating Factor (20 ng/ml), rh Interleukin 3 (20 ng/ml), rh Interleukin 6 (20 ng/ml), rh Granulocyte Colony Stimulating Factor (20 ng/ml) and rh erythropoietin (3 U/ml). The culture was prepared in petri plates (35 mm of diameter) distributing in duplicate 1.1 ml of semisolid ground inoculated. The culture was incubated to 37 °C in 5% CO2 and 95% humidity for 14 days. At term, the determination of the number of colonies was made by microscopic observation classifying the GM-CFU (Granulocyte/Macrophage Colony Forming Units), BFU-E (Erythroid Burst Forming Units), GEMM-CFU (Granulocyte/Erythroid/Macrophage/Megakariocytic Colony Forming Units) and total-CFU in tune with Nissen Druey [27]. Growth was expressed as the clonal efficiency (CE), the ratio between the number of CFU counted and by the number of cells shown in each Petri. The frozen CLP-MT, was thawed in a water bath to 37 °C, diluted 1:4 in ACD-A (Baxter) which is added to with 20 UI/ml of DNase (Sigma–Aldrich) and placed in a centrifuge at 1200 rpm for 10 min. Their pellet was re-suspended in Stemline Hematopoietic Stem Cells Expansion Medium (Sigma–Aldrich) with 15% of ACD-A as well as 40 UI/ml of DNase and then submitted to haemocytometric determination, before execution of the clonogenic test. 2.6. Statistical analysis Data related to the hemocomponent characteristics was expressed as mean and SD. Every step of the procedure was considered as the result of different treatments: concentration (CLP), dilution (CLP-M) and cryoconservation (CLPMT). The significance of variations in the cell counts and fibrinogen concentrations were verified by applying the t-test paired for the comparison of every step with that precedent. Spearman’s correlation coefficient was applied to verify the dependence among concentrations of the cells in the different hemocomponents. The statistically significant level was considered to be p < 0.05. Clonal efficiencies have been submitted to formal statistical analysis (mean, SD, SEM, median, maximum and minimum range) and to the statistic tests of Curtosi, Sweknees and Barlett for control of the normal distribution. Since such tests revealed non-Gaussian distribution of the data, nonparametric methods have been applied (Mann Whitney), using p < 0.05 as the level of significance, for compar-

120

G. Caloprisco et al. / Transfusion and Apheresis Science 42 (2010) 117–124

isons among the clonal efficiencies of the CLP and of the CLP-MT. 3. Results 3.1. Collection and preparation of haemocomponents By using the cell separator, and on average a volume of 1544 ± 191 ml, we obtained a volume of 35 ± 5 ml of CLP and of 185 ± 18 ml of plasma. Subsequently, an average volume of cryoprecipitate of 21 ± 3 ml was obtained from the plasma. Mixing of the three constituents, CLP, cryoprecipitate and DMSO produced an average volume of 41 ± 6 ml of CLP-M. Splitting the CLP-M into eight volumes produced 5.4 ± 1 ml per volume. On average, in every test tube procedure eight activators were obtained; four contained a serum rich in thrombin drawn from whole blood with an average volume of 2.1 ± 0.4 ml each and the other four were of a serum rich in thrombin drawn from the cryoprecipitate with an average volume of 2.9 ± 0.6 ml each. The evaluation of the effectiveness of the activators, measured by the time of clot formation, underlined a significant difference between the two types of thrombin; 45 ± 6 s for the serum drawn from the whole blood and 29 ± 6 s for the serum drawn from cryoprecipitate (p < 0.001). 3.2. Characteristics of haemocomponents Table 1 illustrates the cell composition of the intermediary and final haemocomponents produced with the procedure. The data shows the variations in the cell count due to manipulation of the haemocomponents in the different steps of the procedure: collection with the separator (CLP), mixing with cryoprecipitate and DMSO (CLP-M), and cryoconservation (CLP-MT). The concentration of platelets in the CLP was high, on average, with a very wide range (2052–8196  103/ll) and a factor of enrichment of 16.6 ± 4.2 (range: 8.5–25.6) in comparison with the pre-AP data. Subsequently, dilution with cryoprecipitate and DMSO reduced the concentration of platelets in the CLP-M by 21.8 ± 2.5% on average. The process of freezing and conservation at 80 °C for three months caused a further loss of the platelet’s concentration

in the CLP-MT with a wide range (1100–5138  103 /ll). The average recovery of the platelets in the CLP-MT in comparison with the fresh CLP-M was 81.5 ± 17%. Table 1 also shows an important increase in the CLP of the total WBC concentration with a very wide range (33.5–144  103/ll) and a factor of enrichment of 13 ± 3.3 (range: 4.8–19), in comparison with the pre-AP values. The average decrease of the WBC in the CLP-M, for the process of dilution, was similar to that of platelets (21.2 ± 2.6%). In the CLP-MT the concentration of the total WBC was subsequently reduced with recovery, in comparison with the fresh CLP-M, of 73.6 ± 13%. The count relating to fractions of the total WBC, in particular of the total MN cells and the relative fractions of LY and MO were interesting; specifically, as shown in Table 1, there was a notable increase in the MN in the CLP with a very wide range (29.3–100.8  103/ll) and a factor of enrichment of 28.3 ± 5.5 (range: 13.4–41.1) in comparison with the pre-AP data. Of the MN cells the greater part was represented by the LY, but the MO were well represented also, since they realize a factor of enrichment of 26.5 ± 6 (range: 11.9–40.9) and 35.6 ± 12 (range: 5.3–61.1), respectively, in comparison with the pre-AP values. NE cells were concentrated in the CLP, in comparison with the pre-AP values, with a factor of enrichment of 3.9 ± 2.8 (range: 0.5–13.8), smaller in comparison to the MN cells. In parallel to the total WBC these cell populations, as shown in Table 1, also suffered in the CLP-M and CLP-MT with the decrease of their concentration statistically significant, due respectively to the processes of dilution and cryopreservation. The mean recovery in the CLP-MT, in comparison with the fresh CLP-M, resulted in an MN of 76.8 ± 13.4%, a LY of 79.2 ± 18% and an MO of 79 ± 41%. Fig. 1 shows the relative distributions of the leukocytic cell populations in the donor (pre-AP), and in the haemocomponents produced (CLP, CLP-M, CLP-MT). MN cells increased their share at the expense of the NE share which suffered a notable percentage reduction in the CLP dropping from 58.5 ± 7.9 to 17.2 ± 10.8% (p < 0.001). With rearrangement of distributions there was an important increase in the CLP in particular of the MO share that uses from 8.6 ± 1.7 to 23.7 ± 7.3% (p < 0.001). The increase in the CLP of the LY share that increased from 28.7 ± 7.6% to 58.7 ± 16.2% was also significant (p < 0.001). No significant variation (p < NS) in the percentage acquired in the CLP

Table 1 Cell concentration in donors (pre-AP) and in the hemocomponents during the procedure of collection (CLP), mixing (CLP-M) and cryoconservation (CLP-MT).

PLT (  103/ll) WBC (  103/ll) NE (  103/ll) LY (  103/ll) MO (  103/ll) MN (  103/ll) CD34+ (ll)

pre-AP

CLP

CLP-M

CLP-MT

274 ± 64 6.37 ± 1.7 3.8 ± 1.5 1.75 ± 0.4 0.55 ± 0.18 2.3 ± 0.5 ND

4454 ± 1239* 80.9 ± 24* 15.2 ± 13.3* 45.7 ± 12.5* 18.7 ± 7.2* 64.4 ± 15.2* 81.3 ± 26.7

3482 ± 979** 63.8 ± 19** 12 ± 10.7** 35.7 ± 9.9** 14.6 ± 5.6** 50.3 ± 12.1** ND

2865 ± 1041*** 45.9 ± 13*** 7.1 ± 6.3*** 27.6 ± 7.8*** 10.7 ± 4.9*** 38.2 ± 10.1*** 51.6 ± 19.6**

Data expressed as mean ± SD; PLT, platelets; WBC, total leukocites; NE, neutrophils; LY, linphocytes; MO, monocytes; MN, mononuclear total cells. * p < 0.001 versus Pre-AP. ** p < 0.001 versus CLP. *** p < 0.001 versus CLP-M.

121

G. Caloprisco et al. / Transfusion and Apheresis Science 42 (2010) 117–124 120

180

NE LY MO MN

160 140

CD34+ / microliter

100

120

percent

80

100

60 40

80 60 40 20 0

20

0

20

40

60

80

100

120

3

MN x 10 / microliter

0 pre-AP

CLP

CLP-M

CLP-MT

Fig. 1. Dynamics of the leukocytic percentage composition during the production and cryoconservation procedure of the hemocomponents. NE, neutrophil; LY, lymphocytes; MO, monocytes; MN, mononuclear total cells. Pre-AP, before apheresis collection; CLP, after apheresis collection; CLP-M, after mixing; CLP-MT hemocomponent cryoconservated. Data expressed as mean ± SD.

was shown in the CLP-M or CLP-MT, for all cell populations considered. The RBC counted in the CLP an average of 1.61 ± 0.48  106/ll, with a reduction in the CLP-M to 1.28 ± 0.38  106/ll due to the dilution with cryoprecipitate and DMSO. A further decrement was noticed in the CLP-MT, 1.16 ± 0.35  106/ll, due to the freezing effect. Table 1 also shows the concentration of CD34+ cell counts in the CLP and CLP-MT, on average there were 81.3 cells per ll (range: 18–158) in the CLP and 51.6 cells by per ll (range: 9–106) in the CLP-MT after thawing, with a discrete recovery of 63.7 ± 12% in comparison with the CLP. It must be remembered, however, that the CLP-MT is diluted in comparison with the CLP with addition of the cryoprecipitate and DMSO. The vitality of CD34+ cells counts were respectively 99.4 ± 0.4% in the CLP and 99 ± 0.3% in the CLP-MT. In comparison with the total of MN cells in the CLP the CD34+ represented 0.13 ± 0.04% while, in the CLP-MT it represented 0.14 ± 0.05%. As illustrated in Table 2, the Spearman coefficient shows, for all cell types studied, there was a significant correlation between cell concentrations in the pre-AP control and the CLP control, in particular for the MN cells. Table 2 also shows the correlations between the concentrations of

Table 2 Correlation of the cell concentration before and after collection with cell separator (pre-AP/CLP), and before and after cryoconservation (CLP-M/CLPMT).

PLT WBC NE LY MO MN

Spearman’s coefficient (pre-AP/CLP)

Spearman’s coefficient (CLPM/CLP-MT)

0.58* 0.57* 0.53* 0.57* 0.52* 0.61*

0.85* 0.80* 0.63* 0.57* 0.66* 0.69*

PLT, platelets; WBC, total leukocites; NE, neutrophil; LY, linphocytes; MO, monocytes; MN, mononuclear total cells. * p < 0.001.

Fig. 2. Correlation among the concentrations of the mononuclear cells (MN) and CD34+ progenitors in the CLP, after apheresis collection (r = 0.52; p < 0.001).

cells in the CLP-M and the relative concentrations in the CLP-MT. The Spearman coefficient demonstrated as well that all cell types correlated significantly and in particular the platelets and the total WBC. Fig. 2 shows that there is also a discrete correlation between the concentrations of MN and CD34+ cells in the CLP obtained by apheresis (r = 0.52; p < 0.0001). Less significant is the correlation among the MN and CD34+ cells in the CLP-MT (r = 0.38). The fibrinogen measured in the plasma of the donor was 294 ± 82 mg/dL (range: 206–580) on average, while in the plasma supernatant of the CLP-M it was 720 ± 178 mg/dL (range: 515–1289) realizing an average a factor of enrichment of 2.46 ± 0.1. An elevated correlation was noticed between the concentration of the fibrinogen in the plasma of the donor and in the CLP-M (r = 0.98; p < 0.001). 3.3. Colony forming unit assay In the experiments conducted on the CLP, clonal growth was observed in 20 of the 23 tested samples, one produced CFU sub-confluent and not definable, and two samples did not develop CFU. The results of the formal statistical analysis conducted on the CE of culture of the CLP which proliferated are reported in Table 3. In the CLP-MT, the CFU assays have shown clonal growth in 19 of the 34 tested samples, 3 samples produced CFU which were sub-confluent and not definable, and 12 of 34 did not develop CFU. The results of the formal statistical analysis conducted on the CE of culture of the CLP-MT which proliferated are reported in Table 3. These results reveal that the frequency of the GM-CFU, BFU-E and totalCFU in the CLP-MT are significantly reduced in comparison with that in the CLP (p < 0.05). 4. Discussion With this work we studied the characteristics and the methods to produce a new hemocomponent designed for topical regenerative use on damaged tissue. This hemocomponent is a complex product that was derived in our case from the mixing of a leukocyte platelet concentrated with cryoprecipitate.

122

G. Caloprisco et al. / Transfusion and Apheresis Science 42 (2010) 117–124

Table 3 Entity of the clonal growth in CLP and CLP-MT.

GM-CFU BFU-E GEMM-CFU total-CFU

CLP

CLP-MT

107  10 6 (0–909  10 6) 124  10 6 (0–697  10 6) 44  10 6 (0–252  10 6) 285  10 6 (3–1636  10 6)

0.9  10 4.2  10 0.9  10 5.8  10

6 6 6 6

(0–270  10 6) * (0–297  10 6) * (0–239  10 6) * (0.3–821  10 6)

*

Data expressed as median and range (minimum and maximum) of the clonal efficiency; GM-CFU, Granulocyte/Macrophage Colony Forming Units; BFU-E, Erythroid Burst Forming Units; GEMM-CFU, Granulocyte/Erythroid/Macrophage/Megakariocytic Colony Forming Units; total-CFU, total Colony Forming Units. * p < 0.05 versus CLP.

The platelet, the store of GFs first responsible for the regenerative stimulus, represented the most important constituent of the leucokyte/platelet gel, produced by activation of the CLP-M. Studies on animal models and on man underlined a dose dependent effect between regenerative stimulus and PLT, the concentration of PLT which was most effective seemed to be between 1000–2000  103/ll [3,4]. Our procedure allowed us to produce high concentrations of PLT in the CLP, in comparison with the pre-AP levels, levels which were maintained in the CLP-M after the addition of cryoprecipitate and DMSO. The activation, with the addition of thrombin rich serum in and calcium gluconate, determined a further reduction of about 20% of the platelet concentration in the CLP-M, values that remained at a level of effectiveness, as in the frozen CLP-M. Our procedure of separation has been deliberately modified in order to get a concentrate rich not only in platelets but also in leukocytes. We have studied and applied a protocol of separation capable of, above all, increasing the share of the mononuclear cell’s monocytes. We have achieved a concentration increase in all cell types, particularly the mononuclear population. These cells, as well as the platelets, suffered in the CLP-M and CLP-MT with a significant decrease in concentration due respectively to dilution and to the cryoconservation process, however they remained at acceptable levels. The separation procedure adopted resulted in an important rearrangement of leukocytes in the CLP, in fact, in comparison with the pre-AP data there is a symmetrical inversion in the percentage of the NE and of MN cells in the CLP. MN cells became the most represented population, with an important percentage presence of monocytes. No significant change in the percentage distribution of the cell populations is caused by the dilution and cryoconservation processes in the CLP-M and CLP-MT, showing that cell damage during cryoconservation is proportionally distributed among the various cell types. The choice of a separation procedure that increased the presence of monocytes in the hemocomponent was based on the concept that the tissue macrophage, derived from the circulating monocyte, was a central protagonist in the regulation of the regeneration process. The activated macrophage had a key role in the sequence of events that led to tissue reparation: it starts the inflammatory process, phagocytises the microorganisms, and above all it maintains the proliferation and differentiation processes of cells involved in the repair [13,15]. Therefore, the activation and application of a product rich not only in platelets but also

in monocytes/macrophages should increase the efficiency of the repair process. The optimal cut-off concentration, reported in the literature, for the use of monocytes only in reparation, as in cutaneous ulcerous lesions, was about 2  103 monocytes/microliter [14]. Our procedure allowed us to achieve an average concentration of monocytes about seven times higher in the CLP-M and five times higher in the CLP-MT, so they remained well over effective levels. The activation of platelets concentrated in an injured tissue starts a series of events that involves the resident and hemocomponent cells [12,28]. By applying the activated CLP-M on the injured tissue we brought and facilitated interaction and cooperation between the most important elements of regeneration, platelets, GFs, monocytes, CD34+ progenitors and fibrin, simulating the processes of natural healing [15]. One of the purposes of the adopted procedure was cryopreserving the cells in order to use the hemocomponent for subsequent applications, as in the case of cutaneous chronic ulcers and infiltration tendonitis. To achieve this, a low, final concentration of cryopreservative was used; we noticed, however, the strong recovery in the concentration levels of all the cell types studied in the CLP-MT, when compared with the fresh CLP-M, showing they can maintain sufficient and quality concentration levels in the thawed hemocomponent. Correlations between the concentrations of the pre-AP cells and the CLP cells allowed us to foresee, within certain limits, the level of concentration of a single cell population in every donor. This determined the configuration of the separation procedure which was used to furnish a hemocomponent of predictable composition, with the possibility to estimate what was going to be applied to the injured tissue, all benefit being derived from the quality of the therapy. The fact that even more direct correlations exist between the cell concentrations of the CLP-M and CLP-MT means that the process of cryoconservation determines an approximately linear, and within certain limits, predictable mortality for the same cell population. Also, in the case of cryoconserved hemocomponents there is the possibility, within certain limits, to know the product composition that is applied to the damaged tissue with the benefit of standardization of the regenerative stimulus. The CLP-M activation involved the formation of a gel for transformation of the fibrinogen into fibrin, the biomechanic properties of the gel being the result of the correct fibrin polymerization and stabilization. The fibrinogen concentration for this became a parameter of quality, influenc-

G. Caloprisco et al. / Transfusion and Apheresis Science 42 (2010) 117–124

ing the consistency of the gel which must be manipulable, conformable to the different containers where it is activated, easily reported, sutured as well as hold the tissue involved. These characteristics depend on the amount of cryoprecipitate, the source of fibrinogen. The possibility of varying the volumes of cryoprecipitate in the CLP-M constitution allows one to modify the final cell and fibrinogen concentration and therefore the biomechanical properties of the gel [29,30]. In our case, by constantly maintaining the quota of 15% cryoprecipitate in the CLPM, we reached a compromise between biomechanical characteristics and cell concentrations of the hemocomponent. The gel was activated using serums rich in thrombin produced from whole blood or from cryoprecipitate, the serum from cryoprecipitate was faster in inducing the formation of fibrin, but for our primarily regenerative and non-haemostatic purposes, the serum from whole blood also proved sufficiently rapid. The use of serum rich in thrombin made from cryoprecipitate, produced in a bag, was especially beneficial during surgical operations where rigorous sterility was needed. The adopted procedure of the CLP collection allowed us to obtain, without stimulating the donors with hematopoietic GFs, and by only processing a low haematic volume, a discreet quantity of CD34+ cells. CD34+ cell’s count after three months on the CLP-MT underlined a meaningful decrease in comparison with the CLP; however, the dilution with cryoprecipitate in the CLP involved, for all cells, a reduction of about 20%. The decrease in concentration of the CD34+ in the CLP-MT was the result of two processes: dilution and cryoconservation. The freezing method, with the curve of temperature descent deliberately not controlled allowed a satisfactory final recovery of CD34+ cells. We used this method of freezing to make the procedure of conservation as simple and applicable as possible, even in centres without programmable freezers. The discrete correlation between the MN and CD34+ cells noted in the CLP allowed estimation, within certain limits, of the concentration of CD34+ progenitors collected with the cell separator, confirming the effectiveness and standardization of the separation procedure adopted. The correlation results in the CLP-MT between MN and CD34+ cells were not as helpful, probably because of each cell’s different level of sensitivity to the freezing process, as well as the presence of cell aggregates in the CLP-MT which interfered with flow cytometric counting of the CD34+ cells. This made the CD34+ concentration in the thawed product less accurate. The clonogenic assays on the CLP have underlined that 87% of the hemocomponents tested produced detectable CFU, while only 56% of the CLP-MT produced clonal growth. The fact that a sizeable percentage of the CLP-MT did not produce CFU must be imputed, probably, to the freezing procedure, because the descent of the temperature was not planned; this decision was made deliberately to make cryoconservation with common freezers at 80 °C easily applicable. Data on the clonogenic potential of the CLP and CLP-MT seemed to confirm the observations on the counts of the

123

CD34+, that is, the critical point seemed to be always the process of freezing. It was proved, however, that the CD34+ progenitors, probably involved in the CFU formation, were functional in the CLP and remained such, even if their numbers were diminished in the CLP-MT that did show clonal growth. CD34+ progenitors that were applied on damaged tissue such as bone, tendons and skin seemed to promote vasculogenesis [19–22]. Other studies have shown the importance of cooperation between monocytes/macrophageses, CD34+, and CD133+ circulating cells to induce neovascularization during the tissue reparation [15,31]. With our CLP we particularly wanted to integrate the action of the two cell populations, monocytes/macrophageses and CD34+ to increase the efficiency, with the platelet GFs, of tissue reparation. To this aim we carried out studies on cell culture to verify, in the activated CLP-M, the differentiating orientation of CD34+ cells toward hematopoietic progenitors and also toward mesenchymal progenitors. On this point, recent verifications on the CLP produced with our procedure have underlined the presence of a discrete number of CD133+ cells with mesenchymal orientation (unpublished data, Ferrazza et al., Policlinico Umberto I, Rome University, Italy). In conclusion we were able to affirm that the method of production, with the use of the cell separator, allowed us to realize a hemocomponent that contained the most important cells involved in tissue regeneration. This is an evolution of traditional platelet hemocomponents, which were used for the purpose of regeneration, since it adds to the platelet GF’s stimulus with the presence of other cells, including monocytes, which are involved in the regeneration process. The unexpected return in CD34+, obtained without pre-collection stimulus, and the display of clonogenic potential increase the hemocomponent complexity and enrich the interaction with the tissue. This could be an alternative method, easier and less invasive (in comparison with the collecting of bone marrow) than CD34+ collection for a regenerative product, considering the lower number of manipulations necessary to get the final hemocomponent. Considering that the CLP-M is applied locally on smaller lesions, the number per surface, or unit, volume of CD34+ cells becomes very important. Although the hemocomponent suffers cellular decrements it fundamentally maintains its composition and cell functionality at an effective level. However, the loss in clonal efficiency in the thawed product suggests the necessity, in order to increase the cell functionality, to enhance and eventually modify some parameters of the cryoconservation procedure, preferring, at the moment, the use of a fresh product when possible. The method adopted allows also, changing the relationship of dilution with cryoprecipitate, to modulate the final volume of the hemocomponent based on the cell concentrations, with the possibility of increasing the number of cells. Oversight, standardization and quality control of these hemocomponents are the main points in determining their effectiveness and safety. In the same context, Transfusion Medicine assumes an important role in possessing all the

124

G. Caloprisco et al. / Transfusion and Apheresis Science 42 (2010) 117–124

scientific, technical and legal competencies to use these hemocomponents in routine processes. Appropriate management during all of the production cycles and oversight regarding proper application, through teamwork and with the help of specialists, assures the traceability and safety of the hemocomponent, in a multidisciplinary vision of regenerative medicine. Acknowledgements This work was supported by Regione Veneto, Italy. Project of ‘‘Ricerca Sanitaria Finalizzata” year 2005, n. 222/05. Area: finalized biomedical research. Sector: struggle against the principal diseases and biotechnology for the health. The authors also wish to thank Professors G. Girelli and Dr. G. Ferrazza (SIMT Umberto I, University of Rome, Italy) for helpful discussion and C. Renon (DIMT Belluno, Italy) for technical help.

[13]

[14]

[15]

[16]

[17]

[18]

[19]

References

[20]

[1] Borzini P, Mazzucco L. Platelet gels and releasates. Curr Opin Hematol 2005;12:473–9. [2] Marx RE, Carlson ER, Eichstaedt RM, et al. Platelet-rich plasma: growth factor enhancement for bone grafts. Oral Surg Oral Med Oral Pathol 1998;85:638–46. [3] Grageda E, Lozada JL, Boyne PJ, et al. Bone formation in the maxillary sinus by using platelet rich plasma: an experimental study in sheep. J Oral Implantol 2005;31:2–17. [4] Weibrich G, Hansen T, Kleis W, et al. Effect of platelet concentration in platelet-rich plasma on peri-implant bon regeneration. Bone 2004;34:665–71. [5] Borzini P, Mazzucco L, Panizza R, et al. Randomized trial and local biological effect of autologus platelets used as adjuvant therapy for chronic venus leg ulcers. J Vasc Surg 2004;39:1146–7. [6] Caloprisco G, Barbone E, Borean A, et al. Local haemotherapy of chronic skin ulcers with leukocyte–platelet gel produced from blood components obtained with a cell separator. Blood Transfus 2004;2: 44–54. [7] Caloprisco G, Borean A. Chronic skin ulcers: a regenerative stimulation by topical haemotherapy. Int J Artif Organs 2004;27: 816–7. [8] Mazzucco L, Medici D, Serra M, et al. The use autologus platelet gel to treat difficult-to-heal wounds: a pilot study. Transfusion 2004;44: 1013–8. [9] Virchenko O, Aspemberg P. How can one platelet injection after tendon injury lead to a stronger tendon after 4 weeks. Acta Orthop 2006;77:806–12. [10] Kilian O, Flesch I, Wenisch S, et al. Effects of platelet growth factors on human mesenchymal stem cells and human endothelial cells in vitro. Eur J Med Res 2004;9:1–8. [11] Martineau I, Lacoste E, Gagnon G. Effects of calcium and thrombin on growth factor release from platelet concentrates: kinetics and regulation of endothelial cell proliferation. Biomaterials 2004;25: 4489–502. [12] Giacco F, Perruolo G, D’Agostino E, et al. Thrombin-activated platelets induce proliferation of human skin fibroblasts by

[21]

[22]

[23]

[24]

[25]

[26]

[27] [28] [29]

[30]

[31]

stimulating autocrine production of insulin-like growth factor-1. FASEB J 2006;20(13):2402–4. Danon D, Kovatch MA, Roth GS. Promotion of wound repair in old mice by local injection of monocytes/macrophages. Proc Natl Acad Sci USA Med Sci 1989;86:2018–20. Zulaf-Shani A, Kachel E, Frenkel O, et al. Macrophage suspensions prepared from a blood unit for treatment of refractory human ulcers. Transfus Apher Sci 2004;30:163–7. Anghelina M, Krishnan P, Moldovan L, et al. Monocytes/macrophages cooperate with progenitor cells during neovascularization and tissue repair. Am J Pathol 2006;168:529–41. Cerruti HF, Kerkis I, Kerkis A, et al. Allogenus bone grafts improved by bone marrow stem cells and platelet growth factors: clinical case reports. Artif Organs 2007;31:268–73. Kasten P, Vogel J, Luginbuhl R, et al. Influence of platelet-rich plasma on osteogenic differentiation of mesenchymal stem cells and ectopic bone formation in calcium phosphate ceramics. Cells Tissues Organs 2006;183:68–79. Kocaoemer A, Kern S, Kluter H, et al. Human AB serum and thrombin-activated platelet-rich plasma are suitable alternatives to fetal calf serum for the expansion of mesenchymal stem cells from adipose tissue. Stem cells 2007;24:1270–8. Matsumoto T, Kawamoto A, Kuroda R, et al. Therapautic potential of vasculogenesis and osteogenesis promoted by peripheral blood CD34-positive cells for functional bone healing. AJP 2006;169: 1440–57. Tei K, Matsumoto T, Mifune Y, et al. Administrations of peripheral blood CD34-positive cells contribuite to medial collateral ligament healing via vasculogenesis. Stem Cells 2008;26:819–30. Mifune Y, Matsumoto T, Kawamoto A, et al. Local delivery of granulocite colony stimulating factor-mobilized CD34-positive progenitor cells using bioscaffold for modality of unhealing bone fracture. Stem Cells 2008;26:1395–405. Sivan-Loukianova E, Awad OA, Stepanovic V, et al. CD34+ blood cells accelarate vascularization and healing of diabetic mouse skin wounds. J Vasc Res 2003;40:368–77. Zimmermann R, Jakubietz R, Strasser E, et al. Different preparation methods to obtain platelet components as a source of growth factors for local application. Transfusion 2001;41:1217–23. Council of Europe, Committe of Ministers. In: Strasbourg F, editor. Guide to the use, preparation and quality control of blood components. Recommendation N. R. (95). 6th ed. Council of Europe Publishing; 2000. Galmes A, Besalduch J, Matamoros J, et al. Cryopreservation of hematopoietic progenitor cells with 5-percent dimethyl sulfoxide at 80 °C without rate-controlled freezing. Transfusion 1996;36: 794–7. Miller CL. Human and mouse hematopoietic colony-forming cell assays. From: basic cell culture protocols.. 3rd ed. Totowa, NJ: Humana Press; 2004. Nissen-Druey C. Human hemopoietic colonies in health and disease. 1st ed. Basilea, CH: Karger; 2005. Mogan C, Learson DF. Rationale of platelet gel to augment adaptative remodelling of the injured heart. JECT 2004;36:191–6. Christman KL, Vardanian AJ, Fang Q, et al. Injectable fibrin scaffold improves cell transplant survival, reduces infarct expansion, and induces neovascolature formation in ischemic myocardium. J Am Coll Cardiol 2004;44:654–60. Bensaid W, Triffitt JT, Blanchat C, et al. A biodegradable fibrin scaffold for mesenchymal stem cell transplantation. Biomaterials 2003;24:2497–502. Urbich C, Heeschen C, Aicher A, et al. Relevance of monocytic features for neovascularization capacity of circulating endothelial progenitor cells. Circulation 2003;108:2511–6.