Comparison of proteomic profiles of serum, plasma, and modified media supplements used for cell culture and expansion

A total of 450 mL of whole blood was collected from each of 10 healthy research donors. The blood was collected in two aliquots: one of 375 mL and the other 125 mL. The larger aliquot was collected in CP2D (citrate-phosphate-2dextrose) anticoagulant and was used to prepare plasma. One third of plasma prepared from the CP2D blood was processed to make recalcified plasma, one third to make heat inactivated plasma, and one third was not further processed. The 125 mL aliquot of blood was collected in a bag without anticoagulant and was used to make serum. One and one-half mL aliquots from all components were frozen at -80°C. Aliquots from each of the 10 subjects were tested for 100 soluble factors using a multiplex ELISA. In the first part of the study serum, plasma, recalified plasma and heat inactived plasma were tested. In the second part of the study samples of serum, plasma, and recalcified plasma stored at -80°C were thawed, heat inactivated and tested.

Whole blood was collected using a specially assembled set of blood collection bags consisting of a dry bag from a Pall collection bag system (Leukotrap WB system, CP2D/AS-3 double blood bag unit, Pall Corporation, East Hills, NY 11548, USA) and Baxter, 150 mL transfer bag (Baxter Health Corporation, Fenwal division, Deerfield, IL 60015, USA). The blood collection bags were assembled using a sterile connecting device (SCD 312, Terumo Medical Corp, Elkon, MD). The seven bag set included a phlebotomy needle tubing which was connected to a Y anastomoses tubing with one side connected to a collecting bag labeled (A), which contained 55 mL of CP2D as anticoagulant. Bag A was connected to a dry satellite bag labeled B, which in turn was connected to three 150 mL transfer bags labeled C, D, and E. The second end of Y blood withdrawal tubing was connected to a dry CLX satellite bag (Pall) labeled F, which was connected to second dry bag labeled G.

After 375 mL of whole blood was collected into the first primary bag (A), the tubing leading to bag (F) was then opened and 125 ml of blood was collected into this bag (F) which was placed on ice during phlebotomy. Bags A and F were separated at the end of the collection and the blood in bag A was used to prepare plasma and plasma products, while blood in bag F was used to prepare serum.

To prepare plasma the whole blood in bag A was centrifuged at 4000 rpm for 6.5 minutes (RC3C, Sorval Instruments, Newton, CT). Plasma was expressed into bag B which was then divided equally between the three satellite bags (C, D, and E) with a final volume of 60 to 75 mL in each bag. Plasma bag E was not processed further.

To prepare recalcified plasma calcium chloride (CaCl₂) and thrombin were added to bag C and the plasma was incubated at 37°C for one hour and then stored at 4°C overnight. The bag was then centrifuged at 4000 rpm for 6.5 minutes at room temperature and supernatant, recalcified plasma was expressed into a satellite bag.

Heat inactivated plasma was prepared by incubating plasma in bag D at 56°C for one hour. The bag was then centrifuged at 4000 rpm for 6.5 minutes and the expressed supernatant, heat inactivated plasma, was transferred into satellite bag.

To prepare serum, the whole blood in bag F was stored on ice for one hour. It was then centrifuged at 4000 rpm for 6.5 minutes and the supernatant was transferred into bag G.

Immediately after the preparation of each component was complete, 1.5 mL aliquots were placed into cryovials (1.8 mL Nunc) and snap frozen on dry ice and ethanol. After freezing, all samples were then transferred to -80°C for storage.

The levels of 100 soluble factors were assessed on an ELISA-based platform (Pierce Search Light Proteome Arrays, Boston, MA) consisting of multiplexed assays that measured up to 16 proteins per well in standard 96 well plates[12]. The arrays were produced by spotting 2 × 2, 3 × 3, or 4 × 4 patterns of different monoclonal antibodies into each well of a 96-well plate. Following a typical sandwich ELISA procedure, signal was generated using a chemiluminescent substrate. The light produced at each spot in the array was captured by imaging the entire plate with a commercially available cooled CCD camera. The data was reduced using image analysis software that calculated exact values (pg/mL) based on standard curves. The 100 factors included: interferon alpha (IFN-α), IFN gamma (IFN-γ), interleukin 1 alpha (IL-1α), IL-1β, IL-1Rα, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12p40, IL-12p70, IL-13, IL-15, IL-16, IL-18, tumor necrosis factor alpha (TNF-α), epithelial cell-derived neutrophil activating protein 78 (ENA-78), eotaxin/CCL11, eotaxin II/CCL24, exodus II, growth related oncogene alpha (GRO-α/CXCL1), inducible 309 (I-309/CCL1), interferon inducible protein 10 (IP-10/CXCL10), interferon-inducible T-cell alpha chemoattractant (ITAC/CXCL11), lymphotactin (LTN), monocyte chemoattractant protein 1(MCP-1/CCL2), MCP-2/CCL8, MCP-3/CCL7, MCP-4/CCL13, macrophage-derived chemokine (MDC/CCL22), monokines induced by interferon (MIG/CXCL9), macrophage inflammatory protein 1α (MIP-1α/CCL3), MIP-1β/CCL4, MIP-3α/CCL20, MIP-3β/CCL19, myeloid progenitor inhibitory factor (MPIF-1), neutrophil chemoattractant peptide 2 (NAP-2/CXCL7), stromal-derived factor 1β (SDF-1β/CXCL12), regulated on activation normal T cell-expressed and secreted (RANTES/CXCL5), thymus and activation regulated chemokine (TARC/CCL17), granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), angiogenesis factors, angiopoietin-2 (ANG-2), fibroblast growth factor-α (FGF-α) fibroblast growth factor basic (FGF-b), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), heparin binding epidermal growth factor (HB-EGF), platelet derived growth factor BB (PDGF-BB), vascular endothelial growth factor (VEGF), human growth hormone (HGH), TGF-α, matrix metalloproteinase 1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-10, MMP-13, tissue inhibitor of metalloprotease 1 (TIMP-1), TIMP-2, brain-derived neurotrophic factor (BDNF), nerve growth factor-beta (β-NGF), ciliary neurotrophic factor (CNTF), neurotrophin-3 (NT3), leukemia inhibitory factor (LIF), serum amyloid A (SAA), pregnancy associated plasma protein-A (PAPP-A), amphiregulin (AR), leptin, adiponectin (ACRP-30) adipocytes secreted proteins, Apo-A1, ApoB-100, soluble CD14 (sCD14), CD40 ligand (CD40L), C reactive protein (CRP), myeloperoxidase (MPO), fibrinogen, osteoprotegrin (OPG), osteopontin (OPN), plasminogen activator inhibitor-1 Active (PAI-1), PAI-1Total, aminoterminal proBNP (NT-proBNP), receptor activator of nuclear factor-kappa B (RANK), receptor activator of nuclear factor-kappa B ligand antibodies (RANK-L), vascular cell adhesion molecule (VCAM-1), intracellular adhesion molecule (ICAM-1), L-Selectin, E-Selectin, TNF receptor-1 and 2 (TNFR1, TNFR2), IL-1 receptor (IL-2R), and IL-6R.

Paired two tailed t- test of natural log transformed data was used to compare the plasma and serum soluble factor levels. Differences were considered significant at a cut off p-value of ≤ 0.01. Relatedness of cytokine expression patterns in different types of plasma and serum was tested by applying unsupervised Eisen's hierarchical clustering methods [13] to the data set encompassing the soluble factors across all samples. Unsupervised clustering involved the sorting of both the soluble factors and the samples. In contrast, for unsupervised clustering the factors were sorted, but not the samples. Twenty of the 100 factors were excluded from the analysis since their levels were under the assay's limits of detection. Those factors were: TGF-β, IL-1β, IL-4, IL-5, IL-10, IL-12p70, IL-13, IL-15, IL-17, LIF, NT-proBNP, RANK, RANK-L, PAPPA, MMP-8, B-NGF, VEGF, HB-EGF, IL-3, B-NGF, and FGF-α.

Analysis of the levels of the 80 factors using unsupervised hierarchical clustering among serum, plasma, recalcified plasma components and heat inactivated plasma prepared from 10 subjects yielded three groups of factors. The first group was made up predominantly of heat inactivated plasma components (Figure 1, black bar), the second of serum components (Figure 1, red bar), and the third of plasma and recalcified plasma components (Figure 1, blue bar). The heat inactivated group contained 11 samples: all 10 heat inactivated plasma samples (P-HI-01, P-HI-02, P-HI-03, P-HI-04, P-HI-05, P-HI-06, P-HI-07, P-HI-08, P-HI-09, and P-HI-10) and one recalcified plasma sample (CP-9). The serum group contained 9 serum samples (S-01, S-02, S-03, S-04, S-05, S-06, S-07, S-09, and S-10). The plasma and recalcified plasma group was constituted by 20 samples: 10 plasma (P-01, P-02, P-03, P-04, P-05, P-06, P-07, P-08, P-09, and P-10), 9 recalcified plasma (CP-01, CP-02, CP-03, CP-04, CP-05, CP-06, CP-07, CP-08, and CP-10) and one serum (S-08) samples. The mean levels of all 80 factors are shown in Additional file 1.

The most prominent dissimilarity among the 40 samples was a cluster of 15 factors whose levels were markedly lower in the 10 heat inactivated plasma than in the other samples (Figure 2). These factors were fibrinogen, MIP-1α, MIP-3α, MIP-3β, MDC, MMP-2, MMP-3, MMP-10, ICAM-1, VCAM, SDF-1β, ITAC, MPIF-1, IL-6R, and Ang2.

These results show that plasma and recalcified plasma are very similar in regards to the factors analyzed. However, serum and plasma are quite different as previously reported by our group [14] and heat inactivation affected the levels of several factors.

Previous studies have found that the levels of several factors differed between serum prepared from blood collected in BD vacutainer serum collection tubes and plasma prepared from blood collected in vacutainers with EDTA [14]. A comparison of the soluble factors in the 10 plasma samples prepared in this study from citrated blood collected in bags and the 10 serum samples collected in bags revealed that the levels of 20 factors differed between serum and plasma (Table 1). The levels of fibrinogen and OPN were greater in plasma and levels of 18 factors were greater in serum. Among the other 18 factors whose levels were greater in serum than in plasma were 11 chemokines (MCP-2, MCP-3, MIP-1α, MIP-1β, MIP-3α, NAP-2, ITAC, lymphotactin, eotaxin 2, exodus 2, and RANTES) and 4 factors normally found in platelets NAP-2, RANTES, PDGF-BB, and PAI-1 total.

To produce recalcified plasma thrombin and calcium were added to plasma. As expected, the levels of fibrinogen were lower in recalicified plasma than in plasma, (262 ± 38 microg/mL vs 4,810 ± 1,780 microg/L, p = 0.026). The levels of only 4 other factors differed between recalcified plasma and plasma (MDC, NAP-2, IL-16 and IFN-α) (Table 2). The levels of MDC, IL-16, and IFN-α were greater in plasma and the level of NAP-2 was greater in recalcified plasma.

There were more differences between recalcified plasma and serum than between recalcified plasma and plasma. The levels of 19 factors differed between recalcified plasma and serum (Table 3). Eighteen of the 19 factors were greater in serum and one of the factors was greater in recalcified plasma, OPN. Fourteen of the 19 factors that differed among plasma and serum also differed among recalcified plasma and serum (OPN, MIP-1β, MIP-3α, NAP-2, APO-A1, eotaxin 2, MCP-2, MCP-3, TIMP-1, Lymphotaxin, PDGF-BB, ITAC, PAI-1 total, and TNFR1). The 5 factors that differed among recalcified plasma and serum but not plasma and serum were MDC, MMP-2, L-selectin, APO-B100, and ENA-78.

Heat inactivation was performed on a selected set of supplement samples right after collection to make a preliminary assessment of heat-induced protein levels variation. Plasma was selected as the candidate supplement for testing. A comparison of factor levels between heat inactivated plasma and plasma found that the levels of 36 factors changed with heat inactivation of plasma (Table 4). The levels of 5 factors, MPO, MCP-1, eotaxin 2, MIP-1, PAI-1 total, and TNFR1, were greater in heat inactivated plasma (Table 4). The 31 factors whose levels were greater in plasma than in heat inactivated plasma included several chemokines and growth factors.

Since heat inactivation changed the levels of many plasma factors, stored serum, plasma, and recalified plasma samples from the 10 subjects were heat inactivated and soluble factor levels were measured. A comparison of levels of soluble factors between all heat inactivated and untreated products using supervised hierarchical clustering found that, in general, there were more differences in protein levels between heat inactivated samples and those that were not heat inactivated than among samples that were heat inactivated or among samples that were not (Figure 3). Analysis using t tests found that the levels of 19 factors differed between heat inactivated serum and heat inactivated recalcified plasma (Table 5).