Mesenchymal stromal cells (MSCs) are potent immunoregulators with strong preclinical data that 53 support their application in a wide range of clinical conditions [1] [2] [3] [4] . MSCs can provide therapeutic 54 benefit to patients suffering from systemic inflammation by effectively immunomodulating peripheral 55 blood cells to reduce inflammatory signaling and promote homeostasis. Significant evidence of these 56 MSC derived effects have been shown in vitro, in animal models, and in clinical trials, including recently evaluated, including shifting delivery of the MSCs from intravenous to intramuscular administration, 82 emphasizing higher cell viability, or even using gene modification to promote cell survival [28] [29] [30] [31] . 83 We propose an alternative approach which would minimize direct exposure of the MSCs to blood and 84 contain MSCs in one location. We utilized a recently described perfusion platform that incorporates a 85 hollow-fiber filter into a fluid circuit to compartmentalize the MSCs, while still allowing exchange of 86 signaling molecules from perfusate to cells, and vice-versa [32] . MSCs within this platform were shown 87 to effectively retain their immunomodulatory capacity and alter perfused lymphocyte proliferation, 88 activation, and cytokine production in an MSC dose and duration exposure dependent manner, despite 89 having minimal direct contact with the blood cells. 90
Benchtop coagulation assays, including microfluidic setups, are becoming increasingly translationally 91 relevant [33] . Here, we used a MSC bioreactor platform to assay whether limiting the direct exposure of 92 fresh frozen pooled plasma from healthy patients to MSCs, and therefore the available tissue factor and 93 phosphatidylserine of the MSCs, would affect clot formation time (CFT) in a modified plasma-based clot 94 formation assay [34, 35] . Plasma was perfused through bioreactors seeded with MSCs as well as through 95 circuits in which cells were directly injected into the perfused plasma to make comparative CFT 96 measurements. We were able to show that bioreactor use significantly prolonged CFT relative to direct 97 injection of the MSCs. Flushing of concentrated soluble factors from bioreactors further contributed to 98 prolonged CFTs. Lastly, the previously proposed clinical solution for MSC driven clot formation, 99 anticoagulation with heparin [36] [37] [38] [39] , was shown to be effective in both perfusion setups. These results 100 suggest this new modality for systemic MSC delivery may offer a safer alternative to intravenous MSC 101 injection. The clinically scaled ex vivo engineered MSC delivery is currently undergoing testing in a Phase 102 I/II trial in acute kidney injury (AKI) and COVID-19 associated AKI. 103 MSCs were thawed from cryopreservation into citrated fresh-frozen pooled plasma (FFPP) (George King 116 Bio-Medical, KS, USA) and counted via Trypan Blue exclusion. Each direct injection subgroup was 117 processed individually and subgroups were never combined. The fresh thawed direct injection subgroup 118 was placed into plasma after counting and directly injected for perfusion ( Figure 2 ). The recovered direct 119 injection subgroup allowed for 24 hours of recovery on a cell culture dish, followed by dissociation with 120 TryPLE, counting via Trypan Blue exclusion, and placing into plasma prior to direct injection. Cells for use 121 in washed groups were washed with saline directly after thaw, pelleted and resuspended in fresh 122 citrated media prior to direct injection. 123 124 Bioreactor Fill/Finish MSCs were thawed from cryopreservation into aMEM supplemented with 10% HSA and counted via 126 Trypan Blue exclusion prior to seeding in device. The desired cell number was suspended into 9 mL of 127 aMEM and then seeded into saline-primed microreactors (Spectrum Laboratories, CA, USA; C02-P20U-128 05) with 0, 1, or 3 ×10 6 viable cells per device (0M, 1M, 3M, respectively). Excess media flowed through 129 the semi-permeable hollow fibers while cells remained within the extraluminal space of the reactor. 130
Microreactors used were 20 cm long with an internal surface area of 28 cm 2 . Within each microreactor 131 are nine 0.5 mm diameter fibers comprised of polyethersulfone with a 0.2 μm pore size. The total 132 internal volume of the microreactor is 1.5 mL. 133
Depending on the group, microreactors were either used immediately or incubated at 37℃ for 2 hours 134 to allow for cell attachment and were subsequently held for24 hours at room temperature prior to 135 integration into the circuit. This hold time intends to mimic the time between device manufacture and 136 potential clinical application. 137 
Development of an assay to test CFT under perfusion 218
We developed an approach in which we could test human plasma compatibility of allogeneic MSCs 219 across multiple extents of cell exposure. Cryopreserved MSCs could either be injected into the plasma 220 directly after thaw (Fig. 1A) , cultured for 24 hours after recovery and then directly injected into plasma 221 ( Fig. 1B) , or seeded into hollow-fiber microreactors with a semi-permeable membrane, allowed to 222 attach, and held for up to 24 hours before being subjected to perfusion (Fig. 1C) . These ranges of 223 administration broadly represent many of the systemic administration options available today and allow 224 the comparison of varying degrees of MSC to plasma exposure and the effects of MSC culture 225 conditions. 226
After perfusion, plasma was collected from the circuit via the syringe port and placed into microwells for 227 spectrophotometric reading over time. The point at which a clot was formed was determined by using 228 the resultant spectrophotometric readout and formula (Fig. 1D, E) showed that the presence of MSCs accelerated clot formation under flow conditions relative to acellular 235 controls (Figure 2) . Interestingly, the same experimental setup run with plasma isolated from an individual donor 24 hours after collection instead of FFPP did not result in significantly different clotting 237 times between cellular and acellular groups, likely as it wasn't frozen directly after being pulled (Figure  238   S1 ). Spectrophotometric measurements of optical density at 405 nm captured fibrin clot formation as it 239 occurred within the microwell via increases in measured OD over time (Figure 2A) suggesting that washing to remove debris and cryopreservative did not affect clot time. However, CFT 262 was significantly prolonged by allowing for 24 hours of recovery in culture prior to injection (Figure 3B) . 263
As levels of MSC surface markers appeared to be correlated with clot initiation, we next asked whether 264 limiting the direct interaction of MSC surface markers and plasma could reduce the rate of clot 265 formation. Interestingly, recovery of MSCs within a microreactor did not significantly affect measured 266
CFTs relative to fully recovered MSCs ( Figure 3B ). MSCs that were thawed, seeded into MRs, and 267 immediately perfused, clotted at the same time as MSCs that were seeded and allowed to recover for 24 268 hours. These results suggest that though recovery of cells post thaw significantly reduces MSC induced 269 clotting, housing them in an adherent state on the outside of a hollow fiber seems to further, and more 270 significantly, reduce their clotting potential even without any recovery period. (Figure 6 A) . AMI was induced, animals were re-perfused/stabilized for 1 hour and then connected to 311 the bioreactor perfusion circuit for 12 hours. All animals were perfused without events for 12 hours, 312 with each group showing cardiac injury biomarker induction (Figure 6 B) and similar infarct size (Figure 6  313   C) . Heparin was administered throughout the perfusion process to maintain a minimum activated 314 clotting time (ACT) of at least 300 seconds (as mandated by IACUC), with neither group requiring 315 significantly more heparin than the other (Figure 6 D, E) . These data support Here, in concert with the previously mentioned improvements with cellular production, we assayed the 341 value of incorporating an experimental setup which confines MSCs behind the membrane of a hollow-342 fiber filter. Given that much of the MSCs' ability to induce clot formation arises from its cell surface 343 markers and secreted vesicles, we considered that the confinement of the cells and their procoagulant 344 expressing surface markers in one extraluminal location may reduce the rate at which clot formation 345 occurs. We used an existing bioreactor platform known to retain MSC immunomodulatory capacity in 346 combination with modifications to an existing clot formation assay to assess cellular effect on CFT in this 347 immobilized state relative to direct injection [32, 34, 35] . Through this platform we perfused citrated, 348 platelet poor fresh-frozen pooled plasma. This plasma contains many essential factors for clot initiation, 349
including prothrombin which can be activated with the addition of calcium via CaCl2 to form firm clots 350 over time.
Immediately after CaCl2 addition, this coagulable (but still liquid) plasma is perfused through the hollow-352 fiber filter platform. As expected, direct injection of MSCs into the coagulable plasma perfusion circuit 353 led to rapid clot formation in a dose-dependent manner. Interestingly, perfusion of coagulable plasma 354 through bioreactors seeded with MSCs resulted in clotting at rates significantly slower than their 355 comparable direct injection groups, suggesting that free, circulating MSCs increase thrombosis risk more 356 than bioreactor immobilized MSCs. Like the direct injection group, the MSC dose seeded in the 357 bioreactor was predictive of CFT with higher doses inducing quicker clots, likely through the increased 358 production of pro-coagulable MSC factors. It is also important to note that the presence of a filter 359 Having shown that cell presence shortens clot formation time we sought to use the platform to mitigate 377 that effect as much as possible. Since our microreactor is composed of hollow fibers, exchange does 378 happen through the 0.2 μm pores on the fibers in the microreactor. During hold, MSCs continue to 379 produce materials and some of this accumulated material could be contributing to clot formation. To 380 assess this, we developed a flush protocol and measured steep drops in known coagulation markers. 381
Consequently, flushing resulted in slower CFTs at higher MSC doses. Future studies will assay whether 382 flushing also affects immunomodulatory potential relative to unflushed reactors [32] . 383
Despite the microreactors measured effect of prolonging CFT, it did not completely abrogate the cellular 384 contribution to shortened CFT. In clinical setting anticoagulation protocols will likely be integrated to 385 ensure designated perfusion times are met. Our in vitro experiments and in vivo canine studies showed 386 that heparin administration could effectively prevent any cellular induced clot formation during 387 perfusion. However, many of the patients suffering from systemic inflammation, including those with 388 COVID-19, present with hypercoagulable plasma that will require anticoagulation prior to MSC 389 administration. Our pig model represented a more physiologically relevant condition in which inflamed 390 animals were perfused with a device scaled for human use. Under these acute injury conditions, no 391 clotting was observed in animals perfused with devices loaded with 750M MSCs and for 12 hours. The 392 lack of additional heparin requirement suggests that patients set to undergo MSC-bioreactor perfusion 393 may not need more heparin than a sham control undergoing the same procedure. Future studies 394 comparing direct infusion of MSCs to bioreactor housed MSCs would be useful to evaluate both for 395 coagulation and efficacy responses.
Given the slower CFT in the microreactor groups relative to the direct injection groups, it is possible that 397 a lower dose of heparin could be administered to the microreactor groups. Future dose testing will be 398 required to verify this. Such a finding would be clinically relevant, as reduction in the amount of heparin 399 required to be delivered to critically ill patients may help prevent unintended health consequences. compared to historical controls [57] . The novel delivery approach described here could potentially 413 reduce risk of clot formation from IV administered MSCs, making treatment potentially safer and more 414 controlled than direct infusion. 415
We conclude that immobilization of MSCs in a hollow fiber filter contributes to a reduced clot initiation 417 potential relative to directly injected MSCs. Further removal of cellular byproducts through saline 418 flushing of the bioreactor further reduces the MSC based clot formation potential. Additional heparin does not appear to be required to maintain a designated ACT value relative to acellular perfusion 420 circuits. Taken together, combined integration of these approaches may make MSC therapies which 421 require systemic MSC exposure at less risk for coagulation-related events for a larger population, 422 including the hypercoagulable. The data that support the findings of this study are available from the corresponding author 438 upon reasonable request. 439 440